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The isolation ofinheritance material
• Cell lysis
• Isolation of genom DNA
• Isolation of RNA
• Isolation of plasmid DNA
• Determination of nukleic-acid solution purity and concentration
• based on solution absorbency value
• based on gelelectrophoresis image• DNA- and RNA-based diagnostics and research application
Univ. of Szeged, Med. Biol. Inst., Mol- and cellbiol. pract., VIII.
Genebank Molecular PCR Sequencing Southern-blot Examination of polymorphism: cloning SNP,VNTR, RFLP
White bloodcells
Plant, aninaltissues
Cultivatedcells
Embrionalcells
Forensicsamples
Fossils
Route of gemon DNA isolation from tissues to applications
Tissue sample/cell isolation and cell lysis, making of DNA solution
Isolation of DNA from white blood cells
The sample collection is easy and unexpensive. The sample can be stored easily.
Large amount of genom DNA can be isolated from white blood cells efficiently (1 ml of blood contains 4x106-11x106 white blood cells).
Cell lysis:- Hypotonic treatment - Application of detergent (SDS)
White blood cells: hypotonic shock, detergent
Plant, animal tissues: enzymatic cell wall digest,Homogenizazor with knifes, frozen grinding
DNA isolation from plant, animal tissues, from molds and mushrooms
The intercellular components (plant, mycetes cell wall, fibers of animal connective tissue) makes harder the homogenization or lysis of the cells.
Applied methods:- Enzymatic treatment (digestion of plant, mycetes cell wall).- Desintegration: homogenizator with knives, or glassbeads.- Grinding of liquid nitrogen frozen samples in a mincer.
White blood cells: hypotonic shock, detergent
Tissue sample/cell isolation and cell lysis, making of DNA solution
Cultivated cells: disattachment: trypsin digestionCell lysis: hipotonic shock, detergent
DNA isolation from cultivated cells
The cells form a monolayer in the cell culture flask. Membrane proteins are responsible for cell adhesion.
Disattachment of cells from cell culture flask wall: - Trypsin treatment- Mechanic way
Lysis of the collected cells:- Hypotonic treatment- Use of detergent
From 106 cultivated cells ~ 200μg genom DNA isolated
White blood cells: hypotonic shock, detergent
Plant, animal tissues: enzymatic cell wall digest,Homogenizazor with knifes, frozen grinding
Tissue sample/cell isolation and cell lysis, making of DNA solution
Embryonal cells: differential centrifugation
Can be isolated from amniotic cells.
Embrionic cells
15-20ml amniotic fluids can be gained with amniocentesis. The embrionic cells can be isolated with differential centrifugation from amniotic fluids.
The lysis of cells performed similarly to cultivated cells.
Cultivated cells: disattachment: trypsin digestionCell lysis: hipotonic shock, detergent
White blood cells: hypotonic shock, detergent
Plant, animal tissues: enzymatic cell wall digest,Homogenizazor with knifes, frozen grinding
Tissue sample/cell isolation and cell lysis, making of DNA solution
Forensic sample: small amount of complex samples
Forensic samples
The features of genomic DNS isolation:
- Starting from extremly small amount of cells (eg.: trace amount of cells remained on a cigarette filter).
- Complexity of samples: a.) The isolated cells can be derived from more persons, or from man and animals, too. (eg.: place of dog bite). b.) Physical, chemical and microbiological contamination of the sample. (eg.: dried blood drop on ground).
In most cases the sample collection and genomic DNA isolation needs the consideration of more aspects simultaneously
Embryonal cells: differential centrifugationCan be isolated from amniotic cells.
Cultivated cells: disattachment: trypsin digestionCell lysis: hipotonic shock, detergent
White blood cells: hypotonic shock, detergent
Plant, animal tissues: enzymatic cell wall digest,Homogenizazor with knifes, frozen grinding
Tissue sample/cell isolation and cell lysis, making of DNA solution
Plant, animal tissues: enzymatic cell wall digestion,Homogenisator with knives, frozen grinding
Embrionic cells: Can be isolated from amnion cells differential centrifugation
.
Fossils
The DNA is an incredibly stable macromolecule. Conservated in lifeless, fossilized bones for many million years.
After rubbing to powder the fossils the DNA content can be extracted from the samples.
Fossils: a DNA is an incredibly stable macromolecule, can be rehydrated evenafter millions of years.
Forensic sample: small amount of complex samples
Cultivated cells: Disattachment: trypsin kezeléssel Cell lysis: hypotonic treatment, detergent
White: hypotonic, use of detergent
Tissue sample/cell isolation and cell lysis, making of DNA solution
Genebank Molecular PCR Sequencing Southern-blot Examination of polymorphism: cloning SNP,VNTR, RFLP
White bloodcells
Plant, aninaltissues
Cultivatedcells
Embrionalcells
Forensicsamples
Fossils
Route of gemon DNA isolation from tissues to applications
Cell lysis
+ Chelation
DNA
RNA
Protein
Pronase treatment
Phenolextraction
Genomic DNA isolation with pronase treatment, phenol extraction and precipitation with alcohol
DNA
RNA denat.
proteins
phenol
Alcoholprecipitation
DNARNAprecipitate
Washing
Drying DNA solution
Resolving
+ RNase treatment
Genomic DNA isolation on affinity column
Cell lysis:chaotropic salts eg.: NaIpresence
+Chelating agent+RNase
DNA
Denat.protein
Modified silica-matrix:Binding DNA in presence of chaotropic salts
Denaturated protein
Sample application on silica-matrixcoloumn
Washing Eluationwith low salt conc. solution
DNA solution
DNA
Denat. protein
Addingsilica-matrix coated
magneticbeads
DNA solution
washing
Genomic DNA isolation with magnetic beads
Cell lysis:chaotropic salts eg.: NaIin presence
+Chelating agent+RNase
Eluationwith low salt conc. solution
Easily automatized: Genomic DNA can be isolated from 20 blood samples of 200μl volume within a quarter of hours.
Genomic DNA isolation with magnetic beads
http://www.genomed-dna.com/G_M03_03.htm
http://www.clontech.com/clontech/techinfo/faqs/mn.shtml
http://www1.qiagen.com/
Genomic DNA isolation with pronase treatment, phenol extraction and alcohol precipitation
BioProtocol http://www.bio.com/protocolstools/discipline.jhtml?id=pc1
Genomic DNA isolation with magnetic beadsGenoPrep™ Cartridgewww.genovision.com
Genomic DNA isolation on affinity coloumn
The RNA isolation is based on similar principle as the genomic DNA separation
Characteristics:RNase cannot be inactivated easily. Therefore the contamination must be avoided: application of gloves, RNase free accessories, pipettes, solutions, running system (DEPC treated solution, chaotropic agent). Samples must be kept on low temperature. The purification processes must be performed as quickly as possible.
A few frequently used kits, protocols:Invitrogenhttp://www.invitrogen.comAmbionhttp://www.ambion.com/techlib/basics/rnaisol/index.htmlQiagenhttp://www1.qiagen.com/
Downstreem applications:Northern analysis, RT-PCR, in vitro translation, expression profile determination (DNA chips) and cDNA library construction.
Base of RNA isolation
28S rRNS
18S rRNS
Total RNA specimen
RNA within a strand can produce basepairing, therefore in native condition can take up a spacial form. In order to separate based on molecular size, the 3dimensional form must be distangled. This can be done in denaturation media: heat pretreatment, formaldehide containing media (1%- agarose gel).
Cell lysis:strongly alkalinemedia
+chelatingagent
Plasmid DNA,RNAprecipited
Washing
Drying
Resolving+ RNase treatment
PlasmidDNAsolution
Quick neutralization of solution pH
Deant. genom DNA és denat.protein
Renaturatedplasmid DNA and RNA
Alcoholicprecipitated
+RNA +RNA
Purification of plasmid DNA
Denaturated genome-,plasmid DNA és RNA
Determination of nucleic-acid solution purity
and concentration
• Based on absorbency value of solution
• Based on gelelectrophoresis image
240 260 280 300 (nm)
Ab
sorb
en
cy
RNADNAProtein
A260/A280 > 1.8
Checking nucleic-acid solution purity
Ab
sorb
an
cy a
t 260
nm
Nukleinacid concentration (μg/ml)
2,0
1,5
1,0
0,5
20 40 60 80 100 120
RNADNA
1 A260= 50 μg/ml DNA
1 A260= 40 μg/ml RNA
Calculation of the nucleic-acid solution concentration :
Checking the purity of nucleic-acid solution :
A260/A280 > 1.8
Determination of nucleic-acid solution purity
and concentration
• Based on value of solution absorbency
• Based on gelelectrophoresis image
-
+
Size of DNA molecule :Based on „band” position(circular and linear deviates)
DNS amount:Based on „bands” thickness
~1 μg DNA
Plasmid Linear
Practical:
• Genomic DNA isolation from homogenized pig liver cells
•pUC19 (2686 bp) plasmid isolation Escherichia coli DH5α from a laboratorial bacterial strains
•Determination of nukleicacid solution purity and concentration with gelelektroforezis
Pla
smid
DN
A+
RN
A
Plasmid DNAafter adding RNase
Gen
om
ic D
NA
RNA
Closed ring(supercoiled)
LinearOpen ring
Fragmented chromosomal DNA (linear)
Questions
1, Which substance can not provide DNA during isolation?A .fossils B. human blood C. human red blood cell suspensionD. bacterium colony E. dog hairs
2, Which property of the seen band can provide you information about the molecular amount of DNA during gel electrophoresis?
A. The position B. the „thickness” C. number of bands D. the color E. none of these
3, What is the role of isopropanol during plasmid isolation?A. to denaturate of proteins B. to dissolve DNA molecules C. to remove RNA stains D. to extract nucleic acids from solutionE. to homogenize