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Univ. of Szeged, Med. Biol. Inst., Mol- and cellbiol. pract., VIII.
The isolation of
inheritance 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
Route of gemon DNA isolation from tissues to applications
White blood
cells
Genebank
Plant, aninal
tissues
Molecular
cloning
PCR
Cultivated
cells
Sequencing
Embrional
cells
Southern-blot
Forensic
samples
Fossils
Examination of polymorphism:
SNP,VNTR, RFLP
Tissue sample/cell isolation and cell lysis, making of DNA solution
Isolation of DNA from white blood cells
White blood cells: hypotonic shock, detergent
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)
Tissue sample/cell isolation and cell lysis, making of DNA solution
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.
Tissue sample/cell isolation and cell lysis, making of DNA solution
White blood cells: hypotonic shock, detergent
DNA isolation from cultivated cells
Plant, animal tissues: enzymatic cell wall digest,
Homogenizazor with knifes, frozen grinding
The cells form a monolayer in the cell
culture flask. Membrane proteins are
responsible for cell adhesion.
Cultivated cells:
disattachment: trypsin digestion
Cell lysis: hipotonic shock, detergent
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
Tissue sample/cell isolation and cell lysis, making of DNA solution
White blood cells: hypotonic shock, detergent
Embrionic cells
Plant, animal tissues: enzymatic cell wall digest,
Homogenizazor with knifes, frozen grinding
15-20ml amniotic fluids can be gained with
amniocentesis. The embrionic cells can be
isolated with differential centrifugation
from amniotic fluids.
Cultivated cells:
disattachment: trypsin digestion
Cell lysis: hipotonic shock, detergent
Embryonal cells: differential centrifugation
Can be isolated from amniotic cells.
The lysis of cells performed similarly to
cultivated cells.
Tissue sample/cell isolation and cell lysis, making of DNA solution
White blood cells: hypotonic shock, detergent
Plant, animal tissues: enzymatic cell wall digest,
Homogenizazor with knifes, frozen grinding
Cultivated cells:
disattachment: trypsin digestion
Cell lysis: hipotonic shock, detergent
Embryonal cells: differential centrifugation
Can be isolated from amniotic cells.
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
Tissue sample/cell isolation and cell lysis, making of DNA solution
White: hypotonic, use of detergent
Plant, animal tissues: enzymatic cell wall digestion,
Homogenisator with knives, frozen grinding
Cultivated cells:
Disattachment: trypsin kezeléssel
Cell lysis: hypotonic treatment, detergent
Embrionic cells: Can be isolated from amnion cells
differential centrifugation
.
Forensic sample: small amount of
complex samples
Fossils: a DNA is an incredibly stable
macromolecule, can be rehydrated even
after millions of years.
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.
Route of gemon DNA isolation from tissues to applications
White blood
cells
Genebank
Plant, aninal
tissues
Molecular
cloning
PCR
Cultivated
cells
Embrional
cells
Sequencing
Southern-blot
Forensic
samples
Fossils
Examination of polymorphism:
SNP,VNTR, RFLP
Genomic DNA isolation with pronase treatment, phenol
extraction and precipitation with alcohol
Cell lysis
Pronase
treatment
Phenol
extraction
DNA
RNA
denat.
proteins
Alcohol
precipitation
phenol
+ Chelation
DNA
RNA
precipitate
DNA
RNA
Protein
Washing
Resolving
Drying
+ RNase
treatment
DNA
solution
Genomic DNA isolation on affinity column
Modified silica-matrix:
Binding DNA in presence of chaotropic salts
Cell lysis:
chaotropic
salts eg.: NaI
presence
Sample application
on silicamatrix
coloumn
Washing
Eluation
with low
salt conc.
solution
+Chelating agent
+RNase
DNA
solution
DNA
Denat.
protein
Denaturated protein
Genomic DNA isolation with magnetic beads
Cell lysis:
chaotropic
salts eg.: NaI
in presence
Adding
silicamatrix
coated
washing
Eluation
with low
salt conc.
solution
magnetic
beads
+Chelating agent
+RNase
DNA
solution
DNA
Denat. protein
Genomic DNA isolation with magnetic beads
Easily automatized: Genomic DNA can be isolated from 20 blood samples
of 200μl volume within a quarter of hours.
Genomic DNA isolation with pronase treatment, phenol extraction and
alcohol precipitation
BioProtocol
http://www.bio.com/protocolstools/discipline.jhtml?id=pc1
Genomic DNA isolation on affinity coloumn
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 magnetic beads
GenoPrep™ Cartridge
www.genovision.com
Base of RNA isolation
The RNA isolation is based on similar principle
as the genomic DNA separation
Total RNA specimen
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:
Invitrogen
http://www.invitrogen.com
Ambion
http://www.ambion.com/techlib/basics/rnaisol/
index.html
Qiagen
http://www1.qiagen.com/
Downstreem applications:
Northern analysis, RT-PCR, in vitro translation,
expression profile determination (DNA chips)
and cDNA library construction.
28S rRNS
18S rRNS
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).
Purification of plasmid DNA
Denaturated genome-,
plasmid DNA és RNA
Cell lysis:
strongly
alkaline
media
Renaturated
plasmid DNA and RNA
Alcoholic
precipitated
Quick
neutralization
of solution pH
Washing
Drying
+chelating
agent
Resolving
+ RNase
treatment
Deant. genom
DNA és denat.
protein
+RNA
Plasmid DNA,
RNA
precipited
+RNA
Plasmid
DNA
solution
Determination of nucleic-acid solution purity
and concentration
• Based on absorbency value of solution
• Based on gelelectrophoresis image
Absorbency
RNA
DNA
Protein
240
260
280
300 (nm)
Checking nucleic-acid solution purity
A260/A280 > 1.8
2,0
RNA
DNA
Absorbancy at 260 nm
1,5
1,0
0,5
20
40
60
80
100
Nukleinacid concentration (μg/ml)
120
Checking the purity of nucleic-acid solution :
A260/A280 > 1.8
Calculation of the nucleic-acid solution concentration :
1 A260= 50 μg/ml DNA
1 A260= 40 μg/ml RNA
Determination of nucleic-acid solution purity
and concentration
• Based on value of solution absorbency
• Based on gelelectrophoresis image
-
Plasmid
Size of DNA molecule :
Based on „band” position
(circular and linear deviates)
DNS amount:
Based on „bands” thickness
~1 μg DNA
+
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
Closed ring
(supercoiled)
RNA
Genomic DNA
Plasmid DNA
+RNA
Open ring
Linear
Plasmid DNA
after adding RNase
Fragmented
chromosomal
DNA
(linear)
Questions
1, Which substance can not provide DNA during isolation?
A .fossils B. human blood C. human red blood cell suspension
D. 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 solution
E. to homogenize