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General Techniques for

molecular genetic diagnosisDNA Chemistry

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Nucleic Acids

Deoxyribonucleic acid (DNA)

Ribonucleic acid (RNA)

Polymers made of nucleotides:

base component

sugar component

phosphoryl residue

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Nitrogenous base component

Purine bases

Pyrimidine bases

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Deoxyribose Sugar

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Nucleoside

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Nucleotide

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DNA Backbone

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DNA helix

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Base pairs

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Complementary strands of DNA hydrogen

bond with each other

5'-ATGC-3'

complementary to

5'-GCAT-3' or

3'-TACG-5'

but NOT

5'-TACG-3'

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DNA Helix axis

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Double stranded DNA made up of a

chain and an opposing chain

They can be separated if enough

energy is supplied: Denaturation

They can be joined together again:

hybridization

The chains are annealed

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DNA replication

Because of base pairing acomplementary strand can besynthesized from a single strand

Another strand can be synthesized from

this second strand, and it will beidentical to the initial strand

Replication is carried out by DNApolymerases

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DNA Replication

DNATranscription and

translation

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General Techniques for

molecular genetic diagnosisDNA Purification

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DNA purification

Cell lysis

(e.g. grinding)

Lipids removal

(detergent action)

Proteins digestion,

denaturation

(protease, 2-mercaptoethanol)

DNA precipitation

(ethanol or isopropanol)

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Matrix with positivelycharged groups

DNA spine contains

phosphate groups

negatively charged at pH>2Binding strength in anion-

exchange columns different

between DNA and proteins

(less negatively charged)

Anion-Exchange columns

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Gel Electrophoresis

Method used to identify, purify and

separate DNA fragments

DNA fragments separated according tosize

Fragments 50bp - 20000bp can be well

resolved using various concentrations

of agarose gels

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Electrophoresis is a phenomenon inwhich a charged particle migrates under

the influence of an electric fieldDNA has an ionisable group (PO4

-)

DNA exists as chemically chargedspecies in a solution at a given pH

Under the influence of an electric field, itwill migrate towards the anode

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The process should be carried out in a

buffer to maintain the negative charge

of a DNA molecule (e.g. around 8)Frictional resistance in the gel matrix

makes the fragments to move according

to their respective size

The smallest molecule moves the

fastest and the largest molecule moves

the slowest

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Agarose

Linear polysaccharide

Concentration of agarose controls

pore size of gel

Low concentrations (0.3 -0.8%) =

large pore size

High concentration (1-2.5%) = small

pore size

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Gel running buffer

Tris-buffer variants:1) TAE (Tris-acetate and EDTA): cheap,

fast migration, high resolving power,

but low buffering capacity

2) TBE (Tris-borate and EDTA): higherbuffer capacity, best for low molecular

weight DNA; more expensive, slower

migration, worse for high molecular

weight

3) TPE (Tris-phosphate and EDTA)

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Increase density of the sample

Add colour to the sample Contain negatively charged

dyes (Bromophenol Blue orXylene Cyanol)

Gel loading buffer

DNA ladders

Size marker

Fragments of known size and invarious ranges

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Voltage

Major driving force behind migration

of DNA fragments in the gel

Generally applied : 5-8V/cm

Migration proportional to the appliedvoltage

Southern hybridization: 1-3V/cm

DNA fragment Analysis: 5-8V/cm

Purification of fragments 3-5V/cm

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Detection

Ethidium Bromide (EtBr): intercalates

between the stacked base-pairs of DNAand fluroresces red-orange under UV light

Mutagenic!

Quite sensitive, 10ngDNA bands can be

visualized

Concentration : 0.1 g/mL-0.5 / g/mL

Photographed

SYBR Green (20pg/band) Methylene blue (40 ng/band) without UV

light

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On-line resourcesA Molecular Graphics companion to anIntroductory Course in Biology orBiochemistry. Copyright 1995, Richard B.Hallick.http://www.blc.arizona.edu/molecular_graphics/dna_structure/dna_tutorial.html

Molecular Biology: DNA structure andfunction. http://mcat-review.org/molecular-biology-dna.php

http://molecularhub.com/agarose-gel-electrophoresis/

BookMolecular Biology and Genomics. Mlhardt,C. 2007.Elsevier, London, United Kingdom