Modes of inheritance by pedigree analysis

Genetic disorders are characterized by their patterns of transmission in families.

To establish the pattern of transmission, a usual first step is to obtain information

about the family history of the patient and to summarize the details in the form of

a pedigree (family tree) using standard symbols (figure 1). The patterns shown

by single gene disorders in pedigrees depend basically on two factors:

1. The location of genes on the chromosomes: Autosomal or Sex

chromosomal.

2. The expression of phenotypes: Dominant (expressed even when only one

chromosome of a pair carries the variant allele) or Recessive (expressed

only when both chromosomes of a pair carry the variant allele) mode.

The four basic patterns of Mendelian inheritance are:

Dominant Dominant

Autosomal X-linked

Recessive Recessive

1. Autosomal dominant inheritance

Ex. Marfan Syndrome, Achondroplasia

Every generation shows affected individual.

Vertical transmission is observed.

Both sexes transmit the trait with equal probability.

It gets expressed in both homozygous and heterozygous condition.

Every affected individual has one affected parent.

Sometimes due to variable expressivity and incomplete penetrance (II-

3 or II- 4) there is skipping of generation.

1

2. Autosomal recessive inheritance

Ex: Cystic fibrosis, Phenylketonuria

Horizontal transmission is observed.

Affected people are usually born to unaffected parents.

Males and females get affected with equal probability.

Parents and affected individuals are usually asymptomatic carriers.

After the birth of an affected child, each subsequent child has a 25%

chance of being affected (assuming both parents are carriers and

phenotypically normal).

3. X-linked recessive inheritance

Ex: Haemophilia A, Duchenne Muscular Dystrophy

Affected individuals are usually males.

Affected males are usually born to unaffected parents; the mother is

normally an asymptomatic carrier and may have affected male

relatives

There is no male to male transmission in the pedigree but mating of an

affected male and carrier female can give the appearance of male to

male transmission.

2

4. X-linked dominant inheritance

Ex: Incontinentia pigmenti, Vitamin-D resistant rickets.

Affects either sex, but more females than males.

The child of an affected female, regardless of its sex, has a 50%

chance of being affected.

For an affected male, all his daughters but none of his sons are

affected.

Mitochondrial inheritance

Ex. Diabetes Mellitus with deafness

Genes present on the mitochondria show maternal inheritance.

Variable phenotypic expression of a mutation in mitochondrial DNA

depends on the relative proportions of normal and mutant

mitochondrial DNA.

3

Deviation in the patterns of classical Inheritance patterns

1. Uniparental disomy:

It is defined as the presence of a disomic cell line containing two

chromosomes of a given kind inherited from one parent. Ex.. a female with

cystic fibrosis was found to have two identical copies of most or all of her

maternal chromosome 7.

2. Pseudodominant pedigree patterns:

In case a recessive character is common in the population then the

pedigree pattern resembles the dominant inheritance pattern.

3. Genomic imprinting:

In a considerable number of genetic disorders the expression of the

disease phenotype depends on whether it has been inherited from father

or from mother. This is called genomic imprinting. Ex.Prader willi and

Angelman syndrome.

4. Non- Penetrance:

Some times an occasional skipping of generation is observed in dominant

conditions. A person carrying a mutant gene fails to manifest the disease

but produces affected offspring. This is known as non-penetrance.

5. Anticipation:

A condition in which an inherited disease displays increasing severity

and/or an earlier age of onset in subsequent generations (dynamic

mutation). Ex. Huntington’s disease.

6. Mosaicism

Existence of more than one genetically distinct cell line following a single

fertilization event. Ex. XO / XX.

7. Chimerism

Existence of different cell lines from multiple fertilizations. Ex. XX / XY.

4

Fig 1.The common symbols used in drawing pedigrees

5

Cytogenetic Laboratory Set up

The sterile working place is the most important and basic requirement in the cell

culture laboratories.

The sterility should be maintained in the culture room and cleaned with

antiseptic solutions before starting culture work.

Room and laminar hood should be provided with an ultraviolet light (30

watts), which should be switched on for an hour prior to use. UV light

should be switched off and laminar flow should be on while working in the

hood.

Entry to the culture room should be restricted. Persons using culture room

should wear an autoclaved gown or at least a clean apron.

Contamination of media, sera, phytohaemagglutinin is the greatest hazard

in culture procedure. Care must be taken in the selection and preparation

of materials to be used for tissue culture.

Medium should be prepared under sterile condition under laminar flow. All

glassware/water used should be sterilie.

Table tops and laminar hood should be cleaned with spirit.

Culture room should be steamed or fumigated at least once a month and

must be maintained dust free.

Equipment

Laminar hood

CO2 Incubator

Media filter assembly

Hot air oven

Preparation of Glassware

Prepare 30% acid solution with concentrated HCl or H2SO4 to soak the glassware

overnight. Clean the glassware and transfer to soap water and leave them

overnight. Care must be taken that they are completely dipped in soap or acid

solution. With the help of bottle brush, scrub the glassware thoroughly both inside

6

and outside. Rinse several times under running tap water and then rinse with

distilled water thrice and dry in a hot air oven.

Packing and Sterilization

The dried culture vials with their caps should be packed with aluminium foil and

placed in a glass or aluminium container. The Pasteur pipettes, forceps and

scissors should also be wrapped in aluminum foil individually and packed in

aluminum containers. Keep autoclave tape on the containers or tins. Then

autoclave these containers under a pressure of 15 lbs for 15 minutes (color of the

autoclave tape should changes to black). Transfer the autoclaved stuff into an

oven at a temperature of 60oC for drying.

Preparation of Tissue Culture Media

Reagents

RPMI–1640 nutrient medium, sodium bicarbonate, streptomycin,

gentamycin, sterile double or triple distilled water, Millipore filter.

Apparatus

Filtration assembly with vaccum pump, conical flasks, disposable syringes

and needles.

Prepare media in the culture room using all aseptic precautions. Weigh 10 gms

of RPMI – 1640 nutrient medium and dissolve in one litre of autoclaved triple

distilled water. Add 0.5 ml of streptomycin and 0.5 ml of gentamycin and add

sodium bicarbonate (NaHCO3) and adjust pH 7.0 with 1N HCl or 1N NaOH. Store

the medium at 40C. Filter the media through millipore membrane of 0.22µm and

store it at 40C.

7

Preparation of peripheral blood cells for chromosome analysis

Lymphocytes are differentiated cells, which normally do not undergo subsequent

cell divisions. By culturing lymphocytes in the presence of a mitogen

(phytohaemagglutinin) they are stimulated to replicate their DNA and enter into

mitosis. After an optimum time of the cells being cultured (48 hours for a newborn

and 72 hours for an adult), a mitotic inhibitor, colchicine is added to the

lymphocyte culture. The addition of colchicine to dividing cells acts to prevent the

formation of spindle fibers and, therefore stops mitosis in metaphase. Metaphase

is the optimum phase of mitosis for microscopically visualizing the chromosomes.

By exposing the cells to a hypotonic solution and a series of fixation steps,

metaphase chromosomes can be microscopically observed and analyzed.

Peripheral blood lymphocyte cultures are set up according to modified method of

Moorhead et al (1960) for the detection of karyotypic abnormalities using G-

banding.

Preparation for culture medium and staining

Reagents required

RPMI 1640 medium (Sigma-Aldrich, USA)

Phytohemagglutinin (Sigma-Aldrich, USA)

Fetal bovine serum (Medox, Chennai)

Streptomycin (Sarabhai, Mumbai)

Gentamycin (Fulford, Mumbai)

Sodium chloride (Qualigens, Mumbai)

Potassium chloride (Qualigens, Mumbai)

Giemsa (BDH, Mumbai)

Acetic acid (Qualigens, Mumbai)

Methanol (Qualigens, Mumbai)

Colchicine (Loba Chemie, Mumbai)

Trypsin (Sigma-Aldrich, USA)

8

Preparation of stock medium

RPMI 1640 Lyophilized - l0 g

Triple distilled water - 100 ml

Preparation of phytohemagglutinin (PHA)

M-form is reconstituted with 5 ml sterile triple distilled water

Preparation of antibiotics

Streptomycin - 500 mg

Triple distilled water - 5 ml

Gentamycin - 80 mg

Triple distilled water - 2 ml

Preparation of working medium

RPMI 1640 stock solution - 10.0 ml

Triple distilled water - 90.0 ml

Fetal bovine serum - 20.0 ml

Phytohemagglutinin (M-Form) - 0.5 ml

Streptomycin - 0.25ml

Gentamycin - 0.25ml

pH of the medium is adjusted to 7.2 with 5% Sodium bicarbonate solution. The

prepared medium is stored frozen at -200C until used.

Colchicine solution

a) Stock solution

Colchicine powder - 6 mg

Triple distilled water - 10 ml

b) Working solution

Colchicine stock solution - 0.6 ml

Triple distilled water - 9.4 ml

9

c) Hypotonic solution (O.O75M KCl)

Potassium chloride - 560 mg

Triple distilled water - 100 ml

d) Physiological saline (NaCI 0.9%)

Sodium chloride - 900 mg

Triple distilled water - 100 ml

e) Carnoy's fixative

Methanol and acetic acid in a ratio of 3: I.

f) Giemsa stain

i. Stock solution

Giemsa powder - 1 g

Glycerol - 66 ml

Methanol - 66 ml

To one gram of Giemsa powder 66 ml glycerol is added, gently mixed and

warmed at 600C in a water bath for one hour and cooled to room temperature.

Then 66 ml methanol is added and kept in the refrigerator for fifteen days. It is

then filtered and used as stock.

Working solution

Giemsa stock solution - 2 ml

Phosphate buffer - 2 ml

Triple distilled water - 46 ml

ii. Phosphate buffer ( 0.025M KH2PO4 )

Potassium dihydrogen

ortho-phosphate - 3.4 g

Triple distilled water - 1000 ml (pH 6.8 )

10

Culturing procedure

1. Initiation

1ml of whole blood is obtained after venipuncture into a heparinized

syringe. 0.5ml of the whole blood is added to 5 ml of the working culture

media in duplicates. Cultures are then incubated at 37°C for 72 hours in a

CO2 incubator.

2. Harvesting

Harvesting is carried out by arresting the cells at metaphase by

adding 0.2ml of working colchicine solution at 70th hour, shaken well and

incubated for another two hours. The cultures are then harvested at 72nd

hour as follows:

a) The cultures are gently shaken and transferred to clean

centrifuge tubes and centrifuged at 1500 rpm for 10 minutes.

b) The supernatant is discarded and 5 ml of pre-warmed hypotonic

solution is added to the cell pellet and mixed well. The cell suspension is

incubated for 20 minutes at 37°C. Hypotonic solution is used to swell the

cells for better spreading of the chromosomes.

c) Cell suspension is centrifuged at 1500 rpm for 10 minutes and

the supernatant is discarded. The cells are then fixed in 5ml of freshly

prepared Carnoy's fixative. The cells are kept in the refrigerator overnight

after the first wash with fixative.

d) Later, 3 to 5 washes are given until the cell pellet is clear.

Slide preparation

a) After the final wash, the cells are suspended in 0.5 ml of fresh fixative.

Few drops of the cell suspension are then dropped on to clean pre-chilled

slides.

b) Slides are heat dried at 20°C on a hot plate for applying banding

techniques. Prepared slides are then stored until further use. Slides are

subjected to different staining procedures as follows:

11

Application of Giemsa (G) banding

G banding is carried out by a modified method of Seabright (1971).

Chemicals required

Sodium chloride, trypsin, citric acid, sodium phosphate dibasic

anhydrous, methanol and giemsa.

Preparation of solutions

1) Trypsin solution

Stock solution

Trypsin - 25 mg

Physiological saline - 10 ml

Working solution

Trypsin stock solution (0.25%) - 1 ml

Physiological saline - 49 ml

2) Citric acid (0.1 M)

Citric acid -2.l01g

Triple distilled water -100 ml

3) Sodium phosphate dibasic anhydrous (0.2M)

Na2HPO4 - 2.841 g

Triple distilled water - 100 ml

4) Staining solution

Na2HPO4 - 8 ml

Citric acid - 1.5 ml

Methanol - 1.5 ml

Giemsa - 1 ml

Triple distilled water - 38 ml

12

Procedure

1. Mark the patient’s name, accession number and date on the frosted end of

the slide.

2. Slides should be aged at 600C - 650 C for 2days or at 900C for 1 hour in

the oven.

3. Cool slides to room temperature in covered box.

4. Immerse slides in Trypsin solution. The amount of time varies and should

be adjusted according to age of slide.

5. Immerse slides in Phosphate buffer Saline solution to stop the action of

the trypsin.

6. Place the slide in the coplin jar containing the Giemsa stain for 7 minutes.

The length of time in the stain may vary considerably.

7. Rinse the slides in deionized or distilled water. Wipe the back of the slides.

8. Allow slides to air dry for 10 minutes.

9. Observe under the microscope and look for the banded metaphase

spread for the chromosomal analysis.

Scoring Metaphases

A minimum of 20-25 intact spread metaphses are analysed with image

analyser using Leica software. International System for Human Chromosome

Nomenclature (ISCN 1978) is followed for the construction of karyotypes.

13

Normal female Karyotype showing 46 XX chromosome constitution

14

A schematic representation of Cytogenetic Analysis for screening chromosomal defects.

Normal Male Karyotype showing 46 XY chromosome constitution

Down’s Syndrome showing 47 XY (trisomy 21) chromosome constitution

15

Fluorescence In Situ Hybridization

Molecular cytogenetics is the visualization of sequence specific loci using

biochemical technique of in situ hybridization on cytological preparations. It is

applied to detect chromosomal aberrations in disease conditions. Methods for

non-isotopic labeling and fluorescence detection of nucleic acid probes were

developed in 1980 to overcome the hazardous, complicated and slow procedures

of radioactive DNA probes. The conjugation of reporter molecules like biotin or

digoxigenin to probe nucleotides and detection of such probes using

immunofluorescence technique was the important achievement during the period

1983-1986.

The principle of FISH technique is based on denaturation of the target and

probe DNA sequences and annealing of labeled probe DNA sequences to its

complementary sequences affixed to a glass slide. The selection of probe for

FISH is dependent upon the nature of target sequences to be detected.

16

FISH Probes

Centromeric Probes

Centromeric probes containing alphoid-satellite repetitive DNA sequences

are small insert size clones and are used to detect chromosome copy

number in a cell.

Locus specific probes

Locus specific probes such as cosmids (40kb) or bacteria are used to

detect single gene copy/band specific target sequences.

Whole chromosome painting probes

Whole chromosome painting probes are generated by flow sorted DNA

libraries or by polymerase chain reaction and used for identification of

chromosomes and their rearrangements (translocations/inversions) in a

metaphase.

Equipment / Materials required

Waterbath, shaker incubator, hot plate, microfuge tube,cleaned frosted

glass slides, coverslips (24X24), rubber cement, nail paint.

Reagents required

Sodium chloride, Trisodium citrate, Formamide, Ethanol, NP-40, 4,6,-

diamidino-2-phenylindole (DAPI), 1,4-diazabicyclo[2.2.2]octane (DABCO).

Preparation of reagents

2X SSC buffer

0.882 gm trisodium citrate and 1.753 gm sodium chloride dissolved in 100

ml of distilled water.

Formamide (70%)

35 ml of formamide in 15 ml of 2X SSC buffer

17

Ethanol series

70%,90% and 100%.

Post hybridization washing solution I

49 ml of distilled water and 1ml of 20X SSC buffer and 150µl of NP-40

(derivative of Tween 20) solution.

Post hybridization washing solution II

45 ml of distilled water and 5 ml of 20X SSC buffer and 50 µl of NP-40

solution.

DAPI stain (stock)

2mg/ml (2X SSC) of DAPI

DAPI stain (working)

10µ l of DAPI in 100ml of 2X SSC.

Antifade solution

0.233gm DABCO is dissolved in 800µl of distilled water and add 200µl

of 1M Tris-HCl and 9ml of glycerol.

Method (direct labeled probes)

Slide preparation

1. Metaphase preparations using standard lymphocyte cultures fixed in

carnoy’s fixative are dropped on cleaned frosted slides.

2. The slides are dehydrated with 70%, 90%, 100% ethanol series for 5

minutes in each. (if the cytoplasm is not cleared pepsin treatment is

recommended). The slides are air dried at room temperature.

3. Keep the slides in 70% formamide at 730C in waterbath for 5 min and

denature the metaphases /interphase cells.

4. Transfer the slides into cold ethanol series of 70%, 90%, 100% for 5 min

each.

Probe preparation and denaturation

The probes are prepared according to manufacturer’s instructions

18

1. In a microfuge tube add 1 µl probe diluted with 7µl denaturation buffer

and 2 µl sterile distilled water.

2. When the slides are in 100% cold ethanol, the probe mix is denatured at

730C for 5 minutes

3. The slides are transferred to a hot plate of approximate temperature 450C

4. The denatured probe is added to the marked area on slides and covered

with cover slips with rubber cement or adhesive tape.

5. The slides are kept in slide box (dark box) and incubated at 370C in moist

chamber overnight for hybridization.

6. Remove the coverslips carefully and wash in post hybridization washing

solution- II for 2 min at room temperature and wash the slides in post

hybridization solution -I at 730C in waterbath for 1 minute.

7. The slides are stained with DAPI for 10 minutes.

8. 2 drops of antifade is applied onto the slides and sealed with the cover slip

with nail paint. Press the slide along with coverslip in between the tissue

paper, then apply nail paint along the slides of coverslip.

FISH analysis

The slides are analysed with fluorescence microscope fitted with

CCD camera.

The microscope should have triple band filters for FITC, CY3 or

Texas red or Rhodamine, and DAPI.

Score non-overlapping individual cells or metaphases with intense

bright signals using 10x and 100x magnification.

Do not count cells with overlapping signals in interphase cells.

Cells with diffuse or split signal is considered as one signal.

Use optimum combination of filter set according to fluorochromo

excitation of signal and counter stain.

19

DNA Isolation

DNA isolation is defined as the purification of cellular/ nuclear DNA from

all the components present in the cell. The basic steps of DNA isolation are

disruption of the cellular structure to create a lysate, separation of the soluble

DNA from cell debris and other insoluble material and purification of the DNA

from soluble proteins and other nucleic acids. This is done using organic

extraction (e.g., phenol: chloroform) followed by ethanol precipitation.

DNA can be isolated from all the nucleated cells such as hair, tissue,

blood etc. Certain sources contain high levels of proteins and many types of

secondary metabolites that affect DNA purification. Highly purified DNA is

essential for molecular studies.

Generally DNA is isolated from the human blood using the following two

methods based on classical principles of lysis and purification:

20

i. Phenol Chloroform method

ii. Salting out method

21

Isolation of Genomic DNA

by Phenol Chloroform or Enzymatic Method

The most basic of all procedures in molecular biology is the isolation and

purification of nucleic acids. The key step, the removal of proteins, can often be

carried out simply by extracting aqueous solutions of nucleic acids with phenol,

chloroform and isoamyl alcohol. Additional measures are required when nucleic

acids are purified from complex mixtures of molecules such as cell lysates. In

these cases, it is usual to remove most of the proteins by digestion with

proteolytic enzymes such as proteinase k, which are active against a broad

spectrum of native proteins before extracting with organic solvents.

Digestion of blood sample with Proteinase K will prepare a crude lysate by

digesting cellular protein and SDS is used to break the disulphide bonds. Phenol

is used to remove proteins. Chloroform facilitates the separation of the aqueous

phase and organic phase and iso-amyl alcohol reduces foaming during

extraction. Ethanol helps to precipitate DNA and remove the remaining salts.

Equipment

Refrigerated centrifuge

Water bath

Reagents

1. Erythrocyte lysis buffer

It consists of 155mM or 0.15M Ammonium Chloride, 7.23mM or 0.007M

Potassium Carbonate and 0.5M EDTA dissolved in 1 litre of distilled water.

pH is adjusted to 7.5. It lysis the RBCs.

2. 20% SDS

20grams of sodium dodecyl sulphate is dissolved in 100ml of distilled

water. It is an anionic detergent.

22

3. Proteinase K

20mg of Proteinase K is dissolved in 1ml of autoclaved distilled water.

4. Phenol

Phenol is saturated with equal volume of Tris (pH 7.8) until a pH of 7.8 is

obtained. Liquified Phenol is a clear colourless liquid and can be used for

molecular work without reinstallation. Equilibrated phenol is available with

Banglore Genei.

(Crystalline phenol is not recommended as it must be redistilled at 600C

to remove oxidation products such as Quinine that cause breakdown of

phosphodiester bonds or cause cross-linking of RNA and DNA)

5. 1 M Tris

121.1g of Tris base is dissolved in 800ml of water. The pH is adjusted to

the desired value by adding concentrated HCl. The pH of Tris solutions is

temperature dependent and decreases approximately by 0.03 pH for

every 1oC rise in temperature. The solution is allowed to cool in room

before making final adjustments to the pH. The volume is adjusted to 1

litre with water and sterilized by autoclaving.

6. Chloroform-Isoamylalcohol

Chloroform and Iso-amylalcohol are mixed in 24:1 ratio.

Iso-amylalcohol is frequently used to remove proteins from preparations of

nucleic acid. It also reduces foaming during extraction.

7. 3M Sodium Acetate

408 mg of Sodium acetate is dissolved in 100ml distilled water.

8. Absolute Ethanol

100% Alcohol serves as Absolute Ethanol

23

9. 70% Ethanol

70ml of Ethanol is made upto 100ml with distilled water

10. TE Buffer (pH 8.0)

Tris 10mM 1ml

EDTA 1mM 20µl

PROCEDURE

1. To 5ml of heparinized whole blood add three times equal volume of

erythrocyte lysis buffer. Shake gently and keep it in ice for 15 minutes.

2. Remove tubes from ice and centrifuge in a refrigerated centrifuge at 3,500

rpm for 10 minutes at 4C.

3. Discard supernatant and disturb the pellet with 1 ml of erythrocyte lysis

buffer and make up to 5ml with buffer (repeat the step until white pellet is

obtained).

4. To the sample add 300 µl of 20% SDS and mix gently for 3 to 4 times.

To this mixture, add 40µl of Proteinase K and incubate at 37C in a water

bath overnight.

5. After overnight incubation, add 5ml of phenol mix slowly and centrifuge at

10,000 rpm for 10 minutes.

6. Transfer the supernatant to a fresh autoclaved tube. To this, add 5ml of

Phenol: Chloroform-Isoamylalcohol (25:24:1). Mix gently and then

centrifuge at 10,000 rpm for 10 minutes at 4oC.

7. Transfer the supernatant to a fresh autoclaved tube and add 5 ml

chloroform and Isoamylalcohol (24:1). Mix and centrifuge at 10,000rpm

for 10minutes.

8. Transfer the supernatant to a fresh autoclaved tube and add 3 volumes of

chilled ethanol and keep overnight at -200C. Later centrifuge at 10000rpm

for 10 minutes at 40C.

9. Discard the supernatant and wash the pellet with 70% ethanol and allow it

to dry.

24

10.After the pellet gets dried up, it is dissolved in 200l of TE Buffer and

transferred to 1.5ml eppendorf tube for storage.

Equilibration of Phenol

Before use, phenol must be equilibrated to a pH > 8 because the DNA partitions

into organic phase at acid pH. Store liquefied phenol at -20C. As needed,

remove the phenol from the freezer and then melt it at 68C. Add

hydroxyquinoline to a final concentration of 0.1%. This compound is an

antioxidant, a partial inhibitor or RNase and a weak chelator of metal ions.

To the melted phenol, add an equal volume of Tris buffer (0.5M Tris, pH 8). Stir

the mixture on a magnetic stirrer to 15min. Two phases are formed – upper

aqueous phase removed with the help of glass pipette attached to a vacuum line

equipped with appropriate traps.

Add equal volume of 0.1M Tris to the phenol. Stir the mixture on a magnetic

stirrer for 15 min. Remove the aqueous phase as described in step 2. Repeat

the extraction until the pH of the phenolic phase is >7.8 and store at 40C until

use.

25

2. Isolation of Genomic DNA by “Salting Out” Technique

The DNA extraction using Proteinase K is a lengthy procedure and requires at

least two days. An easy to follow procedure is salting out technique by which

DNA can be extracted within 2-3 hours. In addition to this several kits are

available commercially to isolate DNA within a short period.

The salting out technique has been used successfully to isolate large quantities

of human DNA from whole blood. DNA is extracted from peripheral blood

leucocytes using 5ml of whole blood.

Equipment

Centrifuge

Microcentrifuge

Spectrophotometer

Electrophoresis unit

DC Power supply

Incubator

Reagents

1. TKM 1 buffer (RBC Lysis buffer) 500 ml

Tris HCl (10 mM) pH 7.6 - 0.605 g

KCl (10 mM) - 0.372 g

MgCl2 (10 mM) - 1.016 g

EDTA ( 2mM) - 0.372 g

2. TKM 2 buffer (WBC lysis buffer) 100 ml

Tris HCl (10 mM) pH 7.6 - 0.121g

KCl (10 mM) - 0.074 g

MgCl2 (10 mM) - 1.203 g

EDTA ( 2 mM) - 0.074 g

NaCl ( 0.4 M) - 0.467 g

26

For the preparation of TKM1 and TKM 2 buffers the pH should be maintained by

dissolving Tris in about 70ml. of water and pH is adjusted to 7.6. Now dissolve

EDTA followed by the rest of the chemicals.

3. SDS (10%) - 1gm of Sodiumdodecylsulphate in 10 ml of double distilled

autoclaved water.

4. 6M NaCl - 8.765 gms of NaCl in 25 ml. of distilled water

5. T.E buffer (25 ml)

Tris HCl (10 mM) pH 8.0 – 0.030g

EDTA (1 mM) – 0.609g

Procedure

1. 5 ml of blood is drawn in EDTA vaccutainer, mixed well by inverting the

tube and stored at 4oC till use.

2. Bring the blood sample to room temperature. Then transfer 5 ml of blood

into 15 ml centrifuge tube and add equal volume of TKM1 buffer.

3. Add 100 µl of NP-40 or Triton –X to lyse the red cells. Mix well by

inversion.

4. Centrifuge at 2200 rpm for 10 minutes at room temperature (RT) in a

Beckman table top centrifuge.

5. Slowly pour off the supernatant and save the nuclear pellet (the small

pellet settled at the bottom of the tube) and wash the pellet in 5 ml of

TKM1 buffer and centrifuge as before. Repeat the step if lysis is

incomplete.

6. Add 800µl of TKM2 to the pellet and resuspend the cells.

7. Add 125µl of 10% SDS to lyse the WBCs and mix the whole suspension

thoroughly and incubate for 10 min at 550C. Later transfer the contents

to sterile eppendroff tube.

27

8. Add 300 µl of 6M NaCl to the tube and mix well to precipitate the proteins

by inversion and centrifuge at 12000 rpm for 5 min in a micro centrifuge.

9. Save the supernatant containing DNA and discard the pellet containing

precipitated protein.

10.Transfer the supernatant to a 15 ml tube and add 2 volumes of 100%

ethanol at room temperature. Invert the tube several times slowly till the

DNA precipitates.

11.Remove the precipitated DNA strands with a glass rod and transfer to a

fresh eppendroff tube containing 1 ml of ice cold 70% ethanol.

12.Centrifuge the sample for 5 min at 12000 rpm at 4o C.

13.Dry the pellet in speed vac or in an incubator and resuspend it in 500 µl of

Tris –EDTA buffer at 65oC for 15 min.

ISOLATION OF DNA BY SALTING OUT METHOD

5ml of Blood in 15ml Centrifuge Tube

Add equal volume TKM1 buffer

Add 100µl of Triton X, Mix well by inversion

Centrifuge at 2200 rpm for 10min

Pour off the supernatant to the pellet add 5ml of TKM1 buffer centrifuge as before

Add 800µl TKM2 and resuspend the cells

28

Add 125µl 10% SDS and incubate for 10min at 550C

Add 300µl of 6M NaCl and mix well

Centrifuge at 12000rpm for 5min

To the supernatant, add 2vols of 100% ethanol

Invert the tube several times till DNA precipitates

Transfer the DNA to the fresh eppendorf tube containing 70% ethanol

Centrifuge the sample for 5min, at 12000rpm at 40C

Dry the pellet and resuspend DNA in 500µl of Tris EDTA buffer at 650C for 15min

Estimation of DNA

The isolated DNA needs to be studied for its quality & quantity by using the

following methods.

Spectrophotometric Method

Electrophoretic Method

Spectophotometric Method

1. It is an analytical method used for determining the purity (quality) &

quantity of the isolated DNA.

2. The absorbance is measured at 260nm, at this wavelength an absorbance

of 1.0 corresponds to 50 µg per ml of dsDNA, 40µg/ml of ssDNA or RNA &

20µg/ml of oligo nucleotides.

29

3. The ratio of absorbances at 260nm & 280nm (O.D260/280) provides an

estimation of the purity.

4. For pure DNA and RNA the ratio is approximately 1.8 and 2.0

respectively.

5. If DNA is contaminated with proteins then the ratio will be < 1.8

6. If DNA is contaminated with RNA then the ratio will be > 2.0

Equipment

Spectrophotometer

Reagents

1. DNA sample

2. T.E Buffer

10mM Tris HCl (pH 7.5)

1mM EDTA (pH 8.0)

3. Sterile distilled water

Procedure

1. Add 10µl of isolated DNA sample to 2490µl of T.E buffer / sterile

distilled water (250 fold dilution)

2. Mix well & measure the absorbance at 260nm and 280nm in the

spectrophotometer to determine the quality & quantity of DNA.

3. Calculate the concentration of DNA in the sample by the following

formula:

Quantity of DNA (µg/ml ) = Dilution Factor x Standard(50µg/ml) x O.D at 260nm

30

Calculate the ratio of absorbance at 260nm and 280nm

- If DNA is pure then the ratio will be 1.8-2.0

- If DNA is contaminated with Proteins then the ratio will be <1.8

- If DNA is contaminated with RNA then the ratio will be >2.0

31

Agarose Gel Electrophoresis

The agarose gel electrophoresis is an analytical method basically used for

purification of DNA fragments. Agarose which is extracted from seaweeds, is a

linear polymer of alternating D and L galactose, which are linked by alpha (1-3)

and beta (1-4) linkages. The basic structure is

AGAROSE

Agarose gels are cast by melting the agarose in the presence of the

desired buffer until a clear, transparent solution is achieved. The melted solution

is then poured into a mold and allowed to harden.

Principle

DNA molecules are negatively charged, under electrical field they migrate

through the gel depending on size towards the positively charged electrode and

thus DNA bands can be separated. Migration of DNA molecule through the pores

of the matrix plays an important role in molecular weight separation. The relative

mobility depends upon the concentration and the type of agarose used, the

strength of the applied current, the ionic strength and the density of the DNA

bands separated. Larger molecules move more slowly because of greater

frictional drag and because they worm their drag through the pores of the gel less

efficiently than smaller molecules. Thin gels yield dramatically better results than

thick gels; so cast gels only thick enough to contain the volume of DNA that will

be loaded

Equipment

Gel Electrophoresis unit, moulds, combs, power pack

32

Reagents

10X Tris Borate Buffer (pH 8)

109g Tris

55.6g boric acid

9.3g EDTA

Make up the volume to 1 liter with distilled water.

Procedure

1. Prepare 1X TBE by diluting 10X TBE with double distilled water.

2. Take 40ml of 1X TBE in a 200ml conical flask and add 600mg of agarose.

Boil to dissolve agarose and allow it to cool.

3. Mean while, adjust the combs in electrophoresis set in such a way that

comb on the side is about 2cm from the cathode.

4. When the temperature is around 450C add 2µl of ethidium bromide to

visualise DNA under UV gel doc.

5. Pour the solution slowly into gel tank without creating bubbles.

6. Once the gel gets solidified place the electrophoresis set into the tank and

pour 1X TBE buffer till the level stands 0.5 - 0.8cm above gel surface.

7. Add 5µl of isolated genomic DNA on 2% agarose gel along with the DNA

known amount (Marker DNA) mixed with loading dye.

8. Connect the cathode and anode cords to the power pack, and adjust the

voltage to 60V and run till the dye reaches ¾ of the gel.

9. Then visualize the DNA under the UV gel doc

10.The concentrations of DNA in the unknown sample can be obtained by

comparing the band intensities with the marker DNA.

33

Polyacrylamide Gel Electrophoresis

PAGE is a powerful electrophoretic technique developed to separate

macromolecules on the basis of molecular weight.  The mobility of a molecule in

an electric field is inversely proportional to molecular friction which is the result of

its molecular size and shape, and directly proportional to the voltage and the

charge of the molecule. DNA could be resolved electrophoretically in a semi-solid

matrix strictly on the basis of molecular weight if, at a set voltage, a way could be

found to charge these molecules to the same degree and to the same sign.

Under these conditions, the mobility of the molecules would be simply inversely

proportional to their size. Negatively charged DNA separate within a matrix of

polyacrylamide gel in an electric field according to their molecular weights.

Polyacrylamide is formed by the polymerization of the monomer molecule-

acrylamide crosslinked by N, N’-methylene-bis-acrylamide (abbreviated BIS).

Free radicals generated by ammonium persulfate (APS) and a catalyst acting as

an oxygen scavenger (-N,N,N',N'-tetramethylethylene diamine [TEMED]) are

required to start the polymerization since acrylamide and BIS are nonreactive by

themselves or when mixed together.

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the

polymer chains is dictated by the concentration of acrylamide used, which is

typically between 3.5 and 20%. Polyacrylamide gels are significantly more

annoying to prepare than agarose gels. Because oxygen inhibits the

polymerization process, they must be poured between glass plates (or cylinders).

ACRYLAMIDE

34

The distinct advantage of acrylamide gel systems is that the initial concentrations

of acrylamide and BIS control the hardness and degree of cross linking of the

gel.  The hardness of a gel in turn controls the friction that macromolecules

experience as they move through the gel in an electric field, and therefore affects

the resolution of the components to be separated. Hard gels (12-20%

acrylamide) retard the migration of large molecules more than they do small

ones. In certain cases, high concentration acrylamide gels are so tight that they

exclude large molecules from entering the gel but allow the migration and

resolution of low molecular weight components of a complex mixture.

Alternatively, in a loose gel (4-8% acrylamide), high molecular weight molecules

migrate much farther down the gel and, in some instances, can move right out of

the matrix.

Note: Fragments differing by only few base pairs can be separated better by

polyacrylamide gel.

Equipment

Vertical gel electrophoresis system (Bangalore Genei Pvt.Ltd)

1mm thick combs and spacers

Power pack (capable of maintaining constant voltage of up to 400V)

Reagents

Preparation of 10 % non denaturing Polyacrylamide Gel (29:1)

Acrylamide Solution (30%) - 20 ml

10XTBE - 6 ml

Distilled water - 34 ml

APS (10%) - 400 µl

TEMED - 51 µl

Total volume - 60 ml

35

Preparation of Polyacrylamide Gel Slab

1. Immediately after mixing, pour the gel solutions into apparatus on clean

plates. There should be no trapping of air bubbles.

2. Allow the acrylamide to polymerise at room temperatures for 60 min. The

comb is already inserted in gel before polymerization. When

polymerisation is complete remove the comb.

3. Using disposable micro-pipette and gel loading buffer introduce different

samples in wells formed in the gel.

4. Connect electrophoresis apparatus to power supply with anode and

cathode appropriately joined (The reservoir tanks of electrophoresis

apparatus are filled with 1X TBE buffer).

Running the Gel

1. For polyacrylamide gel electrophoresis pre-run the gel for 30 min with

1V/cm. Then load the samples.

2. Polyacrylamide gels are usually run at a voltage gradient between 1V/cm

and 8V/cm

3. Run the gel until the dye reaches the bottom of the gel. Turn off the power.

The gel is then taken for staining,

4. The reagent most widely used for revealing the positions of DNA bands on

polyacrylamide gels is the fluroscent dye ethidium bromide. This

intercalates into DNA structure and can be seen by its orange/red

fluroscence when excited by UV light.

36

Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is an enzyme catalyzed biochemical reaction in

which small amount of a specific DNA fragment is amplified into large amount of

linear double strand DNA using gene specific oligonucleotide primers. PCR is

commonly used for the detection of hereditary diseases, identification of genetic

fingerprints, diagnosis of infectious diseases, cloning of genes, paternity testing,

and DNA computing etc.

Principle

The DNA is amplified by selecting specific primers. Primers are short oligo

nucleotides usually 18-25 bp that are complementary to the beginning and end of

the DNA fragment to be amplified. They anneal (adhere) to the DNA template at

these starting and ending points, where the DNA-polymerase binds and begins

the synthesis of the new DNA strand using deoxynucleotide phosphates

(dNTPs). The PCR reaction is carried out in a thermal cycler. The PCR process

consists of a series of twenty to thirty-five cycles. Each cycle consists of three

steps.

(1) Denaturation

The double -stranded DNA to be heated to 94-96°C in order to separate

the hydrogen bonds of DNA helix. Prior to the first cycle, the DNA is often

denatured for an extended time to ensure that both the template DNA and

the primers have completely separated and is now single-strand only. The

appropriate time for the reaction is 1 to 5 minutes.

(2) Annealing

After separating the DNA strands, the temperature is lowered so the

primers can attach themselves to the single DNA strands. The

temperature of this stage depends on the primers and is usually 5°C

below their melting temperature (45-60°C). The appropriate time for the

reaction is 1-2 minutes. The melting temperature of a specific oligo

nucleotide primer (Tm) can be calculated by the following simple equation

(3) ExtensionTm = 2 (A+T) + 4(G +C) – 50C.

37

Finally, the DNA-Polymerase has to copy the DNA strands using deoxy

nucleotide tri phosphates (dNTPs). It starts at the annealed primer and works

its way along the DNA strand. The extension temperature depends on the

DNA-Polymerase. The time for this step depends both on the DNA-

Polymerase and on the length of the DNA fragment to be amplified. As a rule-

of-thumb, minute per 1 kbp. A final extension step is frequently used after the

last cycle to ensure that any remaining single stranded DNA is completely

copied. This differs from all other extension steps, only in that it is longer,

typically 10-15 minutes.

Equipment

Thermal cycler

Reagents

The components required are:

DNA template, or cDNA which contains the region of the DNA

fragment to be amplified Two primers, which determine the beginning

and end of the region to be amplified.

Taq polymerase, which copies the region to be amplified.

Nucleotides, from which the DNA-Polymerase builds the new DNA.

Buffer, which provides a suitable chemical environment for the DNA-

Polymerase.

Procedure

To carry out a PCR experiment, the targeted DNA is mixed with a pair of gene

specific oligonucleotide primers, deoxynucleotides and Taq DNA polymerase.

38

PCR reaction mixture (50 µl) consists of the following components

Component Addition order Volume Final concentration

Sterile Distilled Water 1 35.5 µl

10X Reaction Buffer 2 5.0 µl 1 X

dNTPs 3 2.0 µl 200 micro moles

Primer1 4 2.0 µl 20 pico moles

Primer2 5 2.0 µl 20 pico moles

Taq DNA Polymerase 6 0.5 µl 3 units/µl

DNA sample 7 3.0 µl 100 ng

Total mix 50 µl

All the tubes containing 50µl reaction mixture are placed in the thermal cycler.

The cycling conditions of PCR consist of the following steps:

Step 1. Initial denaturation : 95°C for 5 minutes

Step 2. Denaturation : 95°C for 1 minute.

Step 3. Annealing : 65°C for 1 minute.

Step 4. Extension : 72°C for 2 minutes.

Step 5. Steps 2-4 are repeated for 29 cycles.

Step 6. Final Extension : 72°C for 7 minutes.

The amplified products are visualized using 2% agarose gel electrophoresis.

39

Amplification of Refractory Mutation System –

Polymerase Chain Reaction ( ARMS – PCR )

ARMS has been successfully applied in the analysis of a wide range of

polymorphisms, germline mutations and somatic mutations, in the carrier

detection and prenatal diagnosis of inherited diseases. The ability of ARMS to

detect such diseases resides in the discrimination of the technique to selectively

amplify a mutant allele within a vast background of normal alleles. ARMS-PCR is

also known as allele specific amplification (ASA) or PCR amplification of specific

alleles (PASA). ARMS PCR is the quickest and economic form of screening

method available todate.

Principle

A typical ARMS assay comprises two PCR’s, each conducted using the same

template DNA. Both reactions contain a common primer that anneals to an

invariant DNA sequence to one side of the mutation to be detected. The 3’

terminus of the common primer is oriented towards the mutation. In one of the

reactions, the second primer specific for one allele and in the other reaction the

primer is specific for alternative allele.

Primers are designed in such a way that 3’end residue is specific to the mutation

or the normal gene sequence, thus allowing the amplification to occur only when

primer sequence is complementary to the genomic DNA. Genomic DNA is

amplified by using two sets of primers, one internal control set and other specific

for normal / mutant gene.

Equipment

Thermal cycler

Horizontal electrophoretic unit with power pack

40

Reagents

Assay volume - 25µl

Genomic DNA - 0.5 µg

Primers - 0.2µM (each)

dNTPs - 30µM (each)

Taq polymerase - 0.5U

PCR buffer - 1X

Tris HCl - 10mM pH 8.4

KCl - 50mM

MgCl2 - 1.5mM

Thermal Cyling Regimen - 30 cycles

Denaturation - 93oC (1min)

Combined annealing and Extension - 660C - (2 min for first 10 cycles and

1 min for next 20 cycles). Last cycle is extended up to 3min.

41

Amplification for Detection of HbS and HbE Mutations

Amplification for the detection of structural hemoglobins utilizes the same

principle as ARMS PCR.

PCR Mix : Assay volume - 50µl

Genomic DNA - 0.5µg

Primers - 0.5µM (each)

dNTPs - 5µM (each)

Taq polymerase - 1.25U

PCR buffer

Tris HCl -10mM (pH 8.4)

KCl - 50mM

MgCl2 - 1.5mM

Thermal cycling regimen - 30 cycles

Denaturation - 93oC (1min)

Anneling - 68 oC

Extension - 72oC

Last cycle is extended up to 5min.

Primers P7 & P8 along with P14 common primer is used for amplification of HbS

mutation. Primer P9 and P10 along with P14 common primer is used for HbE

mutation. Sample digested with Bhc II or Mnl I.

20 µl of amplified product is analysed by agarose gel elctrophoresis and stained

with ethidium bromide. The gel is visualized on UV transilluminator.

42

Multiplex PCR (M-PCR)

Multiplex PCR (M-PCR) is a technique in which several PCR amplifications are

generated in one PCR reaction with less amount of DNA. This is a rapid, efficient

and less invasive technique to detect the mutations that show deletions.

Multiplex PCR with its simplicity, reliability and cost effectiveness has gained

wider acceptability as a mutation detection method for the diagnosis of DMD and

for identification and distribution frequency of deletions. The deletion information

of the affected individual is necessary for carrier detection. Four sets of primers

which amplify 4 different exons in the DMD gene are used for the identification of

mutations in DMD patients.

Principle

The use of multiple, unique primer sets within a single PCR reaction to produce

amplicons of varying sizes specific to different DNA sequences. By targeting

multiple genes at once, additional information may be gained from a single test

run that otherwise would require several times the reagents and more time to

perform. Annealing temperatures for each of the primer sets must be optimized

to work correctly within a single reaction, and amplicon sizes, i.e., their base pair

length, should be different enough to form distinct bands when visualized by gel

electrophoresis.

PCR Mix - 50µl reaction

250 ng Genomic DNA

20pm each of 4 set of primers

100 µM each of dNTPs

5 U Taq DNA polymerase

10X PCR buffer

25 µl mineral oil (optional)

Final volume 50 µl

43

PCR set up

Initial denaturation 940C for 5 minute which facilitates complete

denaturation of double strand DNA

Followed by 30cycles of denaturation at 940C for 30sec

Annealing of primers to the targeted DNA at 55 0C for 1 minute

Extension of annealed primers at 650C for 2 minutes

Final extension at 650C for 7 minutes

Amplified products resolved by 3% gel electrophoresis stained with ethidium

bromide. The gel is visualized under U.V. transilluminator and documented with

gel-doc system. Sizing of the products is done by running appropriate molecular

weight marker.

44

Molecular Diagnosis of Haemoglobinopathies

by Reverse Dot Blot Technique

Haemoglobinopathies which include the thalassemias and structural hemoglobin

variants like HbS and HbE are a heterogeneous group of recessively inherited

disorders. About 4.5% of the world population carries these abnormal genes. In

India, estimates reveal that there are about 20 million carriers of these defective

genes and around 8000 to 10000 children inheriting a major disease are born

every year.

Globally, about 200 mutations causing thalassemia have been described.

However, each ethnic group has a small number of common mutations and a

larger number of rarer ones. 30 mutations have been described among Indians,

of which 6 mutations are common and account for about 90% of the molecular

lesions.

Principle

The RDB procedure is based on sequence specific oligo nucleotide probes

immobilized on a nylon membrane via linkage of poly T tails and subsequent

hybridization with denatured labeled amplification products that are in solution.

Since the target is not directly bound to the membrane surface, the reaction

kinetics in this assay essentially approach a liquid phase, which allows a rapid

hybridization reaction. If biotinylated probes or biotinylated amplification products

(in the case of the RDB) are used, the non radio reactive detection is usually

carried out with Streptavidin-AP conjugates producing a coloured dot. Under

ideal conditions and in comparison with samples with known concentration, the

colour intensity represents the relative amount of specific amplification products.

45

The following stepwise approach based on PCR and subsequent analyses of the

amplified product can be adopted for molecular characterization and prenatal

diagnosis of the thalassemia syndromes in Indian population.

1. Reverse Dot Blot Hybridization for screening of 5 common thalassemia

mutations [IVSl-5(G-C), IVS11 (G-T), CD8/9(+G), CD41/ 42(-CTIT), CD15

(G-A)] along with HbS and HbE in a single PCR and hybridization step.

2. Detection of the 619 bp deletion by PCR across the breakpoints of the

deletion and electrophoresis.

3. Denaturing Gradient Gel Electrophoresis (DGGE) for screening of rare

and uncharacterized mutations by scanning the entire - globin gene and

its flanking regions in 7 overlapping fragments.

Reverse Dot Blot Hybridization (RDB)

Reagents

1. Membrane - Biodyne C Transfer membrane 0.45µm

2. 2.1- Ethyl-3-(3 Dimethyl amino propyl carbodimide) [EDAC]

3. Blocking agent

4. NBT

5. Streptavidin - AP conjugate

Procedure -Part 1

Processing of Membrane

1. Stamp the Biodyne C membrane

2. Incubate in 0.1 N HCI thrice for 5 min each.

3. Wash the membrane thoroughly with DDW (Change the water thrice)

4. Incubate the membrane in 16% EDC reagent for 15 min. (20mIEDC).

5. Wash the membrane thrice DDW at 5 min interval.

6. Air dry the membrane until it completely dries.

7. Spot the diluted probes on to the membrane

8. Upper row will have normal probes and the lower row will have mutant

probes. Each spot contains 30 picomoles of each probe, diluted in

0.05M carbonate buffer (PH 9.2).

46

9. Air dry the membrane thoroughly.

10. Incubate the membrane for 8 min in 0.1 N NaOH.

11.Wash the membrane thoroughly with DDW.

12.Air dry the membrane thoroughly.

Part - 2. Hybridization and colour development

1. Incubate the blotted membrane in hybridization buffer (2xSSC &

0.1% SDS) for 1/2. hr at 450C

2. Dilute PCR product with 30 l of hybridization buffer and denature

the sample at 95OC for 10min.

3. Add the PCR product to pre-hybridised membrane.

4. Continue hybridization for 1 hr at 450C.

5. In a tray / falcon tube add 50 ml hybridization buffer and incubate at

450C.

6. Take out all the strips from the 15 ml falcons & give a gentle wash

with hybridization buffer in a tray.

7. Transfer all the strips to 50 ml falcon containing hybridization buffer

and washing continued for 15 mins at 45oC

8. Take 10 ml hybridization solution in a tube; add 3.5 l streptavidin

labeled ALP conjugate. This step to be carried out in dark .

9. Pour the solution in a tray and transfer all the strips into the tray

10. Incubate at RT for 1 hr in dark

11. Transfer all the strips into another tray and wash with hybridization

buffer 3 times at intervals of 5 min each

12. Take 12 ml of Buffer C (50 mM Mgcl2, 100 mM Tris pH 9.5, 100 mM

NaCl)

13. Add 200 l substrate solution (NBT /BCIP). This step to be carried

out in dark.

14. Transfer all the strips into a tray and add the substrate prepared

Wait for colour development.

47

Restriction Fragment Length Polymorphism (RFLP)

RFLP is widely used technique to detect known mutations and variations using

specific restriction endonucleases. Genetic defects can be diagnosed by RFLP,

as normal and defective genes give rise to different restriction patterns, if change

is in the recognition site of restriction enzyme.

Principle

Restriction endonucleases recognize specific sequences and cut double strand

DNA within their recognition sequence to produce fragments. Changes in DNA

sequences may generate or abolish or alter the position of recognition site for

restriction endonucleases. Fragments thus produced get separated on agarose

gel electrophoresis and are visualized after staining with ethidium bromide which

enables analysis of sequence variations of discrete region.

Detection of mutations in X-Linked Mental Retardation by RFLP

Mutational analysis in L1 CAM gene causing X-linked mental retardation will be

carried out in male individuals by using RFLP technique. Exon 4 - intron 4

junction of L1 CAM gene (157bp) will be amplified by PCR using specific primers.

Forward Primer: 5’ GGC TCA TGG CCG AGG GTT C 3’

Reverse Primer: 5’ AGG GGA GAA GCT GGG GTG G 3’

Equipment & Reagents

Water bath

Horizontal slab gel electrophoresis apparatus

UV Gel doc

0.2ml PCR tubes

PCR samples

Restriction Enzyme : Taq1

Restriction Buffer (10X)

Distilled Water

Ethidium Bromide

48

Procedure

1. Prepare reaction mixture by adding the following components.

PCR product - 10µl

Autoclaved distilled water - 6µl

10X assay buffer - 2µl

Taq 1 (2 units) - 2µl

Total 20 µl

2. Mix contents of each tube carefully and spin at full speed in a

microfuge for 1 minute.

3. Incubate the mixtures for 2 hours at 650C for digestion of PCR

products.

4. Load the samples in 3% agarose gel and electrophorese for two hours

at 60volts.

5. After electrophoresis stain the gel with ethidium bromide.

6. View the gel under UV gel documentation.

Interpretation

Wild type : After Taq 1 digestion PCR product of 139 bp is seen

Mutant : PCR product of 157bp is seen, indicating absence of Taq1

enzyme site.

Heterozygous : Both 157bp & 139bp fragments are seen in carrier

females.

49

Single Stranded Conformation Polymorphism (SSCP)

Single strand conformation polymorphism (SSCP) technique is preferred among

other mutation detection techniques because it is a rapid and convenient

procedure by which difference in the base pair composition of short DNA strands

can be detected.

Principle

Single stranded DNA has a tendency to fold and form secondary conformation

due to intra molecular bonds at sites of base complementation. These molecules

migrate in non denaturing gels depending not only on the size but also on the

conformation they take up. Any change in the nucleotide sequence affects the

conformation of the molecule which can be detected by shift in the mobility of the

bands on gels.

In this technique PCR amplified products are denatured and snap cooled before

loading onto non-denaturing gel. The electrophoretic mobility of ssDNA depends

on the configuration or shape adopted which is highly variable and sequence

dependent. The detection of sequence variation in DNA is important for the

identification of disease causing mutations in several genetic disorders.

Equipment

Vertical gel electrophoresis system

1mm thick comb and spacers

Power pack (should be capable of maintaining constant voltage of up to

400V)

50

Reagents

1. 10X TBE (pH 8.0)

Tris - 108g

Boric Acid - 54g

0.5M EDTA (pH 8.0) - 40ml

Adjust the pH and makeup the final volume to 1000ml with double distilled

water.

2. Acrylamide Solutions (30%)

Acrylamide - 29g

Bis - acrylamide - 1g

Dissolve in distilled water and make up the volume to 100ml.

3. Ammonium per sulphate (10%)

Ammonium per sulphate - 100mg

Double distilled water - 1ml

Procedure

1. Preparation of 10 % non denaturing Polyacrylamide Gel (29:1)

Acrylamide Solution (30%) - 20 ml

10XTBE - 6 ml

Distilled water - 34 ml

APS (10%) - 400 µl

TEMED - 51 µl

Total volume - 60 ml

Mix the reagents as specified above and pour into the vertical gel mold avoiding

air bubbles. Leave the mold undisturbed for 2 hours for polymerization. After

polymerization is complete, the gel plate is fixed to the unit. Fill the upper and

lower tanks of electrophoresis unit with 1X TBE buffer and pre run the gel for

30minutes at 100V to equilibrate the gel and to maintain the constant

temperature.

51

2. Denaturation of PCR products

Add 5µl of PCR product, 10µl of 95% Formamide and 2µl of loading dye

(0.05% Bromophenol blue; 0.05% xylene cyanol) into 0.2 ml PCR tubes,

mix well and denature at 950C for 5-10 min. Snap freeze in ice to prevent

re-annealing of denatured PCR products.

3. Load 10 µl of denatured samples into the wells of non-denaturing

polyacrylamide gel.

4. Carryout electrophoresis at 200V for about 12-14hrs. The temperature should

be maintained constant throughout the experiment.

5. Visualize the bands by silver staining.

Silver Staining

1. After electrophoresis, separate glass plates carefully to detach the gel and

place the gel in a plastic tray containing distilled water and gently rinse the

gel to remove the buffer.

2. Fix the gel in 10% ethanol by gently shaking for 10 mins.

3. Remove the ethanol by suction.

4. Add 0.7% of nitric acid just enough to cover the gel and gently shake the

tray for 6 mins.

5. Remove nitric acid by suction, add 0.02% of hypo and gently shake for

1min.

6. Rinse the gel in distilled water for 20 sec.

7. Add 0.1% silver nitrate and place in dark for 15mins.

8. Rinse the gel in distilled water for 20 secs.

9. Add 100 ml of developer (Hypo, Na2CO3, HCHO) to the tray.

10.Cover with aluminium foil and shake till the bands develop.

11.To avoid over developing of the gel add 5ml of 10% acetic acid or citric

acid to stop the reaction.

12.Store the gel in distilled water till the documentation.

52

Interpretation

The silver nitrate stained bands are examined for mobility shift in comparison to

control samples. Shift in mobility indicates presence of mutation in the target

region. When 3 or 4 bands are present, it indicates heterozygosity. Exact nature

of mutation can be confirmed further by sequencing.

Parameters affecting SSCP efficiency

1. Size of DNA fragments is the most important parameter to consider in

experimental design. Maximum sensitivity is obtained with small

fragments around 150bp. Smaller DNA fragments are presumably in

capable of forming much secondary structure where as the overall

conformational effect on larger fragments of a single base change may

be too small to give a detectable difference.

2. The temperature at which electrophoresis takes place is an important

factor, since the mobility of SSCPs are heavily dependent on

environmental conditions. It is recommended to carry out

electrophoresis either at room temperature or at 40C conditions.

Unless a laboratory is well air conditioned the ambient temperature will

vary both diurnally and seasonally, thus affecting the reproducibility of

SSCP results.

3. The addition of glycerol in particular has been reported to increase the

sensitivity of SSCP analysis. However, 1mm thick gels containing

glycerol electrophorese very slowly.

4. The ratio of acrylamide cross linking used in SSCP protocols is highly

variable, some groups use a 19:1 ratio others a 29:1 or 49:1. Gels with

a low level of acrylamide : bis-acrylamide cross linking are now thought

to improve the efficiency of SSCP analysis.

53

High Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a form of column

chromatography used frequently in biochemistry and analytical chemistry. It is

also sometimes referred to as high-pressure liquid chromatography.

Principle

HPLC is used to separate components of a mixture by using a variety of

chemical interactions between the substance being analyzed (analyte) and the

chromatography column.

The conventional approach to qualitative and quantitative analyses of

hemoglobin (Hb) molecules for the diagnosis of hemoglobinopathies requires a

combination of tests. An automated HPLC (VARIANT) system can be used to

study alpha-thalassemia and beta-thalassemia syndromes. This automated

HPLC system is an alternative approach to the diagnosis of complicated

thalassemia syndromes.

-Thalassemia is commonly found in the heterozygous state as -thalassemia

trait or carrier. Carrier of the -thalassemia may be affected with mild condition

or may be asymptomatic. Clinical identification of these carriers is important

because, any offspring born to individuals with the -thalassemia trait are at risk

of being homozygous for -thalassemia. The homozygous state; -thalassemia

major is a lethal disease for which there is no adequate treatment. They may

suffer from severe anaemia, jaundice, splenomegaly, bone malformations,

growth retardation and usually death before maturity. Adult blood contains

primarily hemoglobinA (HbA), a small percentage of hemoglobin A2(HbA2) and

trace amounts of fetal hemoglobin(HbF). Carriers of -thalassemia have levels

of HbA2 greater than 3.7% of the total hemoglobin. Determination of A2 and F

levels has become the most practical means to diagnose carriers of -

thalassemia. The Quantitation methods include electrophoresis, anion exchange

54

column chromatography, alkali denaturation etc. High performance liquid

chromatography (HPLC) which is a relatively fast method, has been used for the

determination of various hemoglobins including A2 and F. The VARIANT HPLC

system is fully automated which can be used to separate and determine area

percentage for hemoglobin A2 and F and to provide qualitative determination of

abnormal hemoglobins. The most commonly occurring hemoglobin variants

include hemoglobin D, S, C and E.

This program utilizes the principles of cation-exchange high performance liquid

chromatography (HPLC). Hemolyzed specimens are maintained at 12+2oc in the

automatic sampler chamber. Specimens are sequentially injected into the

analysis stream at 6.5 minute interval. Two dual piston pumps and a pre-

programmed gradient control the elution buffer mixture passing through the

analytical cartridge. The ionic strength of the elution buffer mixture is increased

by raising the percent contribution of elution buffer 2. As the ionic strength of the

mixture increases, more strongly retained hemoglobins elute from the analytical

cartridge. A dual-wavelength filter photometer (415nm and 690nm) monitors

hemoglobin elution from cartridge. Changes in absorbance are monitored and

displayed as a chromatogram of absorbance versus time. Analysis data from the

detector is processed by the built-in integrator and printed on the sample report.

At the end of each sample analysis, a copy of the chromatogram and report data

is automatically printed.

Procedure

A. Preparation of Reagent

1. Elution buffers- readily available with kit.

2. HbA2/F calibrator

Reconstitute the calibrator vial by adding 10ml calibration diluent.

Swirl gently to dissolve and ensure complete mixing. Allow to stand

at room temperature for 10 minutes. Reconstituted calibrator is

stable for 10days at 2-8OC.

55

3. Whole Blood Primer

Prepare the whole blood primer by adding 10ml of de-ionized water

to the vial. Swirl gently to dissolve completely. Allow to stand for 10

minutes at room temperature. Reconstituted whole blood primer will

be stable for 21 days at 2-8OC.

B. Preparation of Sample

Whole blood is collected in a tube with EDTA as anticoagulant. The

samples are stable for 7 days when stored at 2-8OC.

Pipette 5l whole blood to 1.5ml sample vials.

Add 1ml of hemolysis reagent to each sample

Cover each sample vial with parafilm and mix by inversion.

Place the sample vial into VARIANT. Hemolysate is stable for 24

hours when stored at 2-8OC.

C. Run Setup

Prepare patient samples as mentioned before.

Transfer 250l each of primer, de-ionised water and calibrator into

3 sample vials.

Place the reagents and prepared hemolysates into the VARIANT

sample tray as shown below.

STAT Well Hemoglobin primer

Position 1 Deionized water

Position 2 Hb A2 / F calibrator

Position 3-100 Patient hemolysates

Switch on the machine 20 minutes before the procedure. The

system comes to IDLE Status. Then only the procedure can be

started. Enter the programme and IDENTITY code of the samples.

Then START the procedure. Printed Chromatogram will come out

after 6.5 minutes.

56

After completion of the run the system automatically initiates a

WASH cycle for 5 minutes. Then the system enters IDLE status

and the machine can be switched off.

D. Interpretation of Results

The chromatogram can be analysed based on the expected value range.

Patient state HbA2 Level

HbF Level HbS Level HbD Level HbE Level

Heterozygous -Thal

4 to 9% 1 to 5% _ _ _

Homozygous -Thal

Normal or Increased

80-100% _ _ _

Heterozygous HPFH

< 1.5% 10-20% _ _ _

Homozygous HPFH

Absent 100% _ _ _

Heterozygous Sickle Hbs

Normal Normal Increased

Homozygous sickle Hbs

Normal Increased Increased _ _

HbD Trait Normal Normal or Increased

_ Increased _

HbE Trait Increased Normal _ _ _

Thus the VARIANT HPLC System is the easiest method to detect the carrier

status in hemoglobinopathies.

57

Newborn Screening by ELISA

Newborn Screening is basically the practice of testing every newborn for certain

harmful or potentially fatal disorder which is not apparent otherwise at birth. Many

of these are metabolic disorders, often called as inborn errors of Metabolism,

which interfere with the body’s use of nutrients to maintain healthy tissues and

produce energy. Other disorders that may be detected through screening include

problems with hormones, vitamin levels, or the blood. In general, metabolic and

other inherited disorders can hinder an infant’s normal physical and mental

development in a variety of ways. Also, parents can pass along the gene for a

certain disorder without even knowing that they are carriers.

With a simple blood test in which blood sample is taken by a heel prick from the

foot of the baby, doctors can tell whether newborns have certain conditions that

could eventually cause problems. Even though these conditions are considered

rare and most babies are given a clean bill of health, early diagnosis and proper

treatment can make the difference between lifelong impairment and healthy

development.

Universal screening for metabolic disorders today is mandatory in US, Europe

and many countries in both North and South Asia. No such screening is currently

available in India. The pilot programme has been initiated at the Institute of

Genetics, to provide screening for treatable disorders by early detection.

Congenital Hypothyroidism (CH) is the most commonly found disorder in India

that affects 1 in 5000 babies worldwide and is seen predominantly in females

than in males. CH affected babies do not have enough thyroid hormone and

therefore develop symptoms such as mental retardation, poor growth, deafness,

neurological abnormalities, cretinism and developmental delay. This disorder can

be detected immediately after birth and can be treated with oral doses of thyroid

hormone to attain normal development.

58

Similarly Congenital Adrenal Hyperplasia (CAH) also can be treated by

supplementing the deficient hormone. Over production of androgens causing a

salt crisis and virilisation in females and acidosis leading to cardiovascular

collapse are commonly observed symptoms in patients with CAH. Screening for

these disorders significantly reduces the mortality and morbidity associated with

them.

Neonatal Thyroid Stimulating Hormone (TSH) Screening

The Neonatal TSH Screening is a quantitative assay for the determination of

Thyroid Stimulating Hormone (TSH, Thyrotropin) for congenital hypothyroidism

(CH). The hypothalamus secretes Thyrotropin releasing hormone (TRH) which

stimulates the release of TSH by the pituitary gland. Thyroid Stimulating

Hormone (TSH) is a 28.3 Kda glycoprotein which is carried by the bloodstream to

the thyroid gland where it stimulates the synthesis and secretion of the thyroid

hormones triiodothyronine (T3) and thyroxine (T4).

Circulating concentrations of TSH are regulated through a feedback control

system involving the hypothalamus, pituitary and thyroid glands and also by a

negative feedback mechanism involving the direct action of T3 and T4 on the

pituitary. Thus when thyroid hormone concentrations are increased as in

hyperthyroidism, TSH secretion is inhibited. Conversely, when thyroid

concentrations are decreased as in hypothyroidism, TSH secretion is stimulated.

Congenital Hypothyroidism (CH) can result in mental retardation which can be

prevented by early treatment. The most effective method of assessing neonatal

thyroid function is a combination of T4 and TSH screening. Confirmation of

hypothyroidism should be performed by using serum T3, T4 and TSH

determinations prior to initiating therapy.

59

Prinicple

Neonatal TSH Screening is a sandwich Enzyme Linked Immuno Sorbent Assay

(ELISA). The sample is incubated with a peroxidase-labelled anti-TSH

monoclonal antibody (Conjugate Reagent) in a microwell coated with another

anti-TSH monoclonal antibody. After incubation, the wells are washed free of

unbound labelled antibody. TSH in the sample is determined by the reaction of

the bound conjugate with substrate producing a coloured product. The

concentration of TSH in the sample is proportional to the colour measured at

450nm in a plate reader. TSH concentrations are expressed as μIU/ml whole

blood.

Reagents

1 Coated Reaction Wells Wells coated with monoclonal anti-TSH antibody2 Conjugate Reagent Enzyme labelled anti-TSH antibody solution

(labelled with Horse-Raddish Peroxidase)3 Substrate Reagent TMB4 Stop Solution Reaction stop solution (1% sulphuric acid).5 Wash Buffer

Concentrate20 X Wash Buffer

6 Blood Spot Standards Whole Blood containing approximately 5, 10,

30 and 65 μ IU/ml. TSH spotted & dried on S & S 903 paper.

7 Blood Spot Controls Whole Blood containing approximately 3,7

& 30 μ IU/ml. TSH spotted & dried on S&S

903 paper.

8 Plate sealer plate sealing sheets

Specimen Collection and Handling

Blood should ideally be collected in circle approximately one half inch in diameter

on S & S 903 specimen collection paper. These samples should be obtained

from a heel prick of a 3 – 5 day old infant. Blood should completely fill the pre-

marked circle on the collection card. The card should be allowed to dry

completely at room temperature and kept away from direct sun light, dust,

moisture and heat. Specimens should be shipped in a sealed paper envelope at

60

room temperature. Upon receipt, specimen can be stored at 2 – 8˚C in a

moisture–proof environment for up to 14 days. Care should be taken to assure

uniformity of blood spot punching. Spots should be punched from similar areas

and away from fingers and printed marks. Spots showing evidence of pooling,

clotting or incomplete saturation should be rejected. Specific rejection criteria for

questionable blood spots are the responsibility of the screening laboratory. Cord

blood should not be used as a specimen.

Preparation of reagents and storage

All the reagents in kit are ready to use except for the wash buffer.

Store all reagents in the QuantaseTM Neonatal TSH Screening Assay kit at

2 – 8˚C until kit expiration.

Unused reaction strips should be stored in the pouch sealed with

dessicant.

Wash Buffer: Prepare the wash buffer by mixing one bottle of 20X

concentrated Wash Buffer concentrate (100 ml) with 1900 ml of deionized

water. The diluted (1X) Wash Buffer is stable for 60 days at room

temperature (18 – 25˚C).

Note

Each laboratory should establish its own normal range and cutoff concentrations

to account for variations in patient populations. Values in the assay are

expressed in μIU/ml blood. The factor for converting μIU/ml whole blood to

μIU/ml serum is 2.22.

Procedure

The standards, controls and patient specimens are eluted directly in the coated

reaction wells with the Conjugate Reagent.

1. Punch 1 x ⅛ inch diameter disc from each standard and control or patient

specimen into a clean well of the coated reaction plate. Standards should

be punched in duplicate.

61

2. Add 100 μl of conjugate reagent into all wells containing standards,

controls and patient specimens.

3. Seal the wells. Place the micro titer plate on a shaker and shake at room

temperature (18 – 25˚C) for 3½ hours at 350 rpm. Ensure all punched

discs are immersed in the Conjugate Reagent throughout the shaking.

4. Decant the contents of the wells, including the punched discs into a

suitable container. Wash the plate with a plate washer, or manually, 5

times using a minimum of 300μl Wash Solution per well. After the last

wash, firmly tap the plate on absorbent paper to remove any residual

wash solution.

5. Add 100 μl of Substrate Reagent to all wells. Seal the wells with a Plate

Sealer and incubate the plate at room temperature (18 – 25˚C) for 30

minutes in dark.

6. Add 100 μl of Stop Solution to all wells. Gently mix the plate.

7. Read the plate at 450nm within 30 minutes.

Interpretation of Results

The recommended screening ranges are: Normal : 0 – 10 μIU/ml

Borderline : 10 - 20 μIU/ml

Abnormal : > 20 μIU/ml

62

Neonatal 17 - OHP Screening

The Neonatal 17 -OHP Screening is a quantitative assay for the determination of

17 – α Hydroxyprogesterone (17 – OHP) in Congenital Adrenal hyperplasia

(CAH). CAH, a recessively inherited defect results from any of the enzymatic

steps required to synthesize cortisol from cholesterol. Persistently high

concentrations of 17–OHP due to the blockage of the pathway, converting the

precursor steroids to cortisol, are considered presumptively diagnosis of CAH

resulting from 21–hydroxylase deficiency. Complete or partial deficiency of 21–

hydroxylase accounts for 90 – 95% of all CAH cases.

Principle

Neonatal 17 – OHP Screening is a competitive enzyme-immunoasay (EIA). Dried

Blood Spot Specimens are eluted directly into anti-rabbit IgG antibody coated

microwells in a solution containing peroxidase-labelled 17–OHP and anti-17-OHP

antibody. After incubation, the wells are washed free of unbound labelled 17-

OHP and antibody. Bound 17–OHP in the sample is determined by the reaction

with bound peroxidase labelled 17-OHP and substrate producing coloured

product. Because of competition between 17–OHP in the specimen and enzyme

labelled 17–OHP, the colour generated is inversely proportional to the

concentration of 17–OHP in the specimen measured at optical density 450nm in

a plate reader. 17–OHP concentrations are expressed as ng/ml whole blood.

Reagents

1 Coated Reaction Wells Wells coated with monoclonal anti-rabbit IgG antibody

2 Conjugate Reagent Enzyme labelled 17 – OHP solution (labelled with Horse-Radish Peroxidase)

3 Antibody solution Anti – 17 – OHP antibody solution4 Substrate Reagent TMB5 Stop Solution Reaction stop solution (1% sulphuric acid).6 Wash Buffer 20 X Wash Buffer

63

Concentrate7 Blood Spot Standards Whole Human Blood containing approximately 0,

5, 10, 20, 50 and 100 ng/ml. 17 - OHP spotted &

dried on S&S 903 paper.

8 Blood Spot Controls Whole Human Blood containing approximately

7.5 & 40 ng/ml.

17 - OHP spotted & dried on S & S 903 paper

9 Plate sealer plate sealing sheets

Specimen Collection and Handling

Blood should ideally be collected in circle approximately one half inch in

diameter on S&S 903 specimen collection paper. These samples should be

obtained from a heel prick of a 3 – 5 day old infant. Blood should completely fill

the pre-marked circle on the collection card. The card should be allowed to dry

completely at room temperature away from direct sunlight, dust, moisture and

heat. Specimens should be shipped in a sealed paper envelope at room

temperature. Upon receipt, specimen can be stored at 2 – 80C in moisture –

proof environment for up to 14 days. Care should be taken to assure uniformity of

blood spot punching. Spots should be punched from similar areas and away from

fingers and printed marks. Spots showing evidence of pooling, clotting or

incomplete saturation should be rejected. Specific rejection criteria for

questionable blood spots are the responsibility of the screening laboratory.

Preparation of reagents and storage

All the reagents in kit are ready to use except for the wash buffer.

Store all reagents in the QuantaseTM Neonatal 17 - OHP Screening Assay

kit at 2 – 8˚C.

Unused reaction strips should be stored in the pouch sealed with

desiccant.

64

Wash Buffer

Prepare the wash buffer by mixing 1 bottle of 20X concentrated wash

buffer concentrate (100 mL) with 1900 mL of deionized water. The diluted

(1X) wash buffer is stable for 60 days at room temperature (18 – 25˚C).

Note

Each laboratory should establish its own normal range and cutoff concentrations

to account for variations in patient populations. Values in the assay are

expressed in ng/ml blood. The factor for converting μIU/ml whole blood to ng/ml

serum is 2.22. The factor for converting ng/ml whole blood to nmol/L whole blood

is: 0.33 ng/ml whole blood = 1 nmol/L whole blood

PROCEDURE

1. The standards, controls and patient specimens are eluted directly into

the coated wells with the conjugate Reagent.

2. Punch 1 x ⅛ inch diameter disc from each Standard and Control or

patient specimen into a clean well of the coated reaction plate.

Standards should be punched in duplicate.

3. Add 50 μl of conjugate reagent into all wells containing Standards,

Controls and patient specimens.

4. Add 50 μl of Antibody Solution to each well.

5. Seal the wells. Place the microtitre plate on a shaker and shake at

room temperature (18˚C – 25˚C) for 3½ hours at 350 rpm. Ensure all

punched discs are immersed in the Conjugate Reagent throughout the

shaking.

6. Decant the contents of the wells, including the punched discs into a

suitable container. Wash the plate with a plate washer, or manually, 5

times using a minimum of 300 μL Wash Solution per well. After the last

wash, firmly tap the plate on absorbent paper to remove any residual

wash solution.

65

7. Add 100 μl of Substrate Reagent to all wells. Seal the wells with a

Plate Sealer and incubate the plate at room temperature (18–25˚C) for

30 minutes in dark.

8. Add 100 μl of Stop Solution to all the wells and gently mix the plate.

9. Read the plate at 450nm within 30 minutes.

Interpretation of Results

The recommended screening ranges are : Normal : < 65 ng/ml

Borderline : 65 - 89 ng/ml

Abnormal : > 90 ng/ml

66

Comet Assay

The comet assay is a simple, rapid and sensitive technique for analysing and

quantifying DNA damage in individual mammalian cells. This was first introduced

by Ostling and Johanson in 1984. The image obtained looked like a “comet” with

a distinct head, comprising of intact DNA and a tail. The more versatile alkaline

method of the comet assay was developed by N.P. Singh and co-workers in

1988. This method was developed to measure low levels of strand breaks with

high sensitivity.

Principle

In the comet assay, the cells are embedded in a thin agarose gel on a

microscope slide. The cells are lysed to remove all cellular proteins, and the DNA

is subsequently allowed to unwind under alkaline conditions. Following unwinding

the DNA is electrophoresed and stained with silver nitrate. During

electrophoresis, broken DNA fragments (damaged DNA) or relaxed chromatin

migrates away from the nucleus. The extent of DNA liberated from the head of

the comet is directly proportional to the DNA damage.

Equipment

1. Electrophoresis unit

2. Power pack

3. Microscope slides

Preparation of Reagents

1. Lysing Solution (stock)

36.52gm of NaCl, 9.3 gm of EDTA, 0.3 gm of Tris are dissolved in 100 ml

of distilled water and stirred for 15min on the magnetic stirrer. 3gm of

sodium hydroxide is added to the mixture and allowed to dissolve. The pH

is adjusted to 10 with HCl or NaOH and the volume is made up to 250 ml.

The solution is stored at room temperature.

67

2. Lysing Solution (Working)

1% Triton-x and 10% DMSO is added to the stock lysing solution (89ml) to

make it up to 100ml and refrigerated for 30 minutes prior to use (pH 10).

3. Electrophoresis buffer

12 gms of NaOH, 0.36 gms of EDTA are dissolved in 1litre of distilled

water (pH –13).

3. Neutralizing buffer

48.5 gm of Tris is dissolved in 1000ml of distilled water to make 0.4M Tris

buffer (pH 7.5).

4. Fixing solution

75 gm of TCA, 25 gm of ZnSO4, 25gm of glycerol are added in 500 ml of

distilled water.

5. Silver Staining Solution (stock)

Solution A

25g of Sodium Carbonate in 500 ml of water.

Solution B

100mg of Ammonium Nitrate, 100 mg of Silver Nitrate, 500mg of

Tungstosilicic acid and 250 l of 37% Formaldehyde are dissolved

in 500 ml of distilled water.

6. Staining solution (Working)

Just before use add 32 ml of solution A and 68 ml of solution B and

pour the mixture over the dried gel slides.

7. Stopping solution

1% Glacial Acetic Acid.

68

Methodology

1. Preparation of pre-coated slides

Clear plain slides are dipped in hot 1%NMPA and one side is wiped. The

slides are dried over night at 370c. These slides can be used for three

weeks.

2. Layering of gels

Pre coated slides are layered with a mixture of 110l of 0.5% LMPA and

20l of whole blood. The cover slip is placed and gel is allowed to set for 4

to 10 minutes on ice pack. The cover slip is slided off and then the slides

are layered with 110l of LMPA for the third layer. The cover slip is placed

on the gel and allowed to set for 4 –10 minutes on ice pack. The cover slip

is slided off.

3. Lysing of the cells

The slides are placed in cold lysing solution at 40C for 1 – 24 hours.

4. Unwinding of DNA

The slides are taken from lysing solution and placed side by side in

horizontal electrophoresis unit. The electrophoretic chamber is filled with

alkaline buffer until the buffer level completely covers the slides. Bubble

formation is avoided. The slides are kept in buffer for 20 min to allow the

DNA for unwinding.

5. Electrophoresis

Turn on the power supply to 25 volts and current is adjusted to 300mA by

raising or lowering the buffer level. Electrophoresis is carried out for 20

min and the slides are lifted gently and placed in a tray.

6. Neutralizing the slides

The slides are flooded with 0.4M Tris neutralizing buffer for 10 min and the

buffer is drained off and the step is repeated twice.

7. Fixing the slides

69

The slides are placed in fixing solution for 10 minutes and washed for

several times with distilled water. The slides are air dried at room

temperature overnight.

8. Staining the slides

Fresh staining solution is poured over the slides uniformly. They are gently

shaked for 20-30 minutes. Then the slides are dipped in a jar containing

stopping solution for five minutes until yellowish brown color is developed.

The slides are washed with distilled water and dried in inclined position at

room temperature. The slides can be stored in dust free environment for a

long period.

9. Screening the slides

The slides are screened under microscope for comets and the tail lengths

are measured using occulometer.

10. Interpretation

DNA damage is estimated based on the tail length and percentage of

comets.

70

Good Laboratory Practices (GLP)

There are some fundamentals of laboratory work such as handling of samples

and how to behave in the laboratory. It is well known that laboratories are not the

most healthy work environment. The presence of volatile hazardous organic

liquids, explosive chemicals and laboratory equipment do not guarantee optimal

conditions for a safe and healthy working place. Nevertheless, it is possible to

organize the laboratory environment in such a way, that normal work is possible

without health problems. To manage safe, healthy and trouble free environment

in the laboratory some common rules should be followed by the analysts and

attendants in the laboratories. Good Laboratory Practices (GLP) are as follows..

Safety Aspects

All laboratory staff should be well versed and responsible for proper

functioning of all the safety equipments and personal protective wares.

Do not keep more chemicals in the laboratory than necessary for the

ongoing work. Store the rest in a safe place.

Always wear laboratory coats and safety glasses. Eating, drinking and

smoking should be prohibited in the laboratory.

Ensure that safety devices and precaution manuals are easy to find.

Take care of fire extinguishers, fume hoods, chemical spill kit, eye

washes and other safety devices. Always have a first-aid kit ready in

the laboratory.

Used liquids/chemicals should be disposed off in a proper way. Follow

the procedure precisely.

Check analytical procedures if they are not clear, especially while

working with organic liquids which may be dangerous.

Be careful with power supply, gas cylinders and heating equipments.

Work as much as possible in a fume hood and always add acid/base to

water.

71

Don’t try to catch falling glassware. Remove and replace broken

glassware.

Laboratory staff should undergo an annual medical examination.

Safety procedures should be displayed as poster in the laboratory.

Some training for laboratory staff is necessary for handling

emergencies.

Laboratory Hazards

Contact with chemicals may cause external or internal injuries.

External injuries are caused by exposure to caustic/corrosive

chemicals (acid/base/reactive salts). Prevent as far as possible

inadvertent spills and splashes and equipment corrosion.

Internal injuries may result from toxic or corrosive effects of chemicals

accidently ingested and absorbed by body.

Inorganic acids and bases have health and safety limits. Exposure to

fumes can irritate or damage eye, skin and create respiratory

problems. Hot acids quickly react with the skin.

Store acids and bases separately, in well-ventilated areas and away

from volatile organic and oxidisable substances.

Slowly add strong acids and bases to water to avoid spattering. If there

is an accidental skin contact, thoroughly flush the contaminated area

with water and seek medical attention.

Perchloric acid reacts violently or explosively on contact with organic

materials.

Don’t use perchloric acid together with organic reagents, particularly

volatile solvents in one fume hood.

NaOH and certain other chemicals produce considerable heat on

dissolution, which may cause burns.

Some metals (arsenic, nickel, mercury) are highly toxic and may also

be carcinogenic. Avoid inhalation, ingestion and skin contact.

72

Nearly all organic solvents are hazardous. Some are probably

carcinogenic and should be treated with extra caution.

Avoid mouth pipetting. Use of auto pipettes is advisable.

Beware of physical hazards from electrical items and gas cylinders.

Always remember, HOT glassware looks exactly like COLD glassware,

be careful while handling.

Preparing for the lab

The student - laboratory plays an important role in giving the student a

"hands on" opportunity to verify chemical principles and learn important

techniques for safe chemical manipulation. In order to get the most out

of the laboratory work, simple suggestions have been enlisted below.

Carefully read the laboratory experiment and any suggested additional

reading (s) before coming to lab.

Do the assigned pre-lab exercises (if any). These generally cover any

calculations or important observations which need to be made.

Make a list of questions regarding the experiment and get it clarified.

This can save hours of wasted time in the laboratory.

All data should be recorded in an appropriately bound log book. Do not

use loose sheets or ring binders, as the loose sheets can be easily

lost.

Make a brief outline of the experiment including calculations for

needed reagents/solutions in your notebook for a quick start.

Prepare data recording format ahead of time. Well prepared data

recording format not only speeds up the recording of data, but also

helps greatly during calculations and report writing.

Clean your glassware at the end of the lab period so that it is ready for

the next laboratory work.

Many people forget to write down their observations. Colour changes,

endothermic or exothermic changes, changes in physical state, boiling

73

point, melting point, freezing point, etc are very crucial to record. Look

at the data, do they look reasonable for the type of experiment and

expected results? When in doubt, repeat a portion of the experiment,

there is no better lesson than to find your own mistake. If you are still

unsure, ask the lab instructor.

Lab instructor will sometime discuss the important aspects of the lab

with students individually or in small groups in an effort to help them

get more out of the experiment.

Lastly, it is important to follow the safety do's and don'ts of the

laboratory, not only for your own safety but also for your fellow beings.

74