Download The genetic material

SZTE, Orv. Biol. Int., Mol- és Sejtbiol. Gyak., VIII.
The genetic material
The genetic material is always nucleic acid(DNA,RNA)
(1)Griffith, (2)Avery, MacLeod and McCarty
(3)Hershey and Chase
This genetic material contains mayority of the information necessary for
life
Understanding the genetic material leads us to understand how beings –
including us – „work” and
we gain the possibility to cure genetic diseases by mending „errors” of the
genetic material
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Isolation of genetic material
White blood
cells
Plant, aninal
tissues
Cultivated
cells
Embrional
cells
Forensic
samples
1, Disclose the sample
2, Separate nucleic acids from
other macromolecules
3, Experimental and
diagnostical uses
Genebank Molecular cloning PCR Sequencing
Southern-blot
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Polymorphism
Fossils
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)
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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.
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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
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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.
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.
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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
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Tissue sample/cell isolation and cell lysis, making of DNA solution
White: hypotonic, use of detergent
Fossils
The DNA is an incredibly stable
macromolecule. Conservated in lifeless,
fossilized bones for many million years.
Cultivated cells:
Disattachment: trypsin kezeléssel
Cell lysis: hypotonic treatment, detergent
After rubbing to powder the fossils the
DNA content can be extracted from the
samples.
Forensic sample: small amount of
complex samples
Fossils: a DNA is an incredibly stable
macromolecule, can be rehydrated even
after millions of years.
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Univ. of Szeged, Med. Biol. Inst., Mol- and cellbiol. pract., VII.
The isolation of
genetic material
1, Disclosing the sample – cell lysis
2, Separation of macromolecules(proteins nucleic acids etc.)
found in the sample by physical-chemical properties
3, Cleaning of nucleic acids
4, Determining the purity, concentration and quality of isolated
nucleic acids by
• UV absorpion of the solution
• picture of gel-electrophoresis
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Genomic DNA isolation with pronase treatment, phenol
extraction and precipitation with alcohol
1, Cell lysis
2,
Proteinase
treatment
2, Phenol
extraction
DNA
RNA
denat.
proteins
3, Alcohol
precipitation
phenol
+ Chelation
DNA
RNA
precipitate
DNA
RNA
Protein
3, Washing
Drying
Resolving
+ RNase
treatment
DNA
solution
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Genomic DNA isolation on affinity column
Modified silica-matrix:
Binding DNA in presence of chaotropic salts
1, Cell lysis:
chaotropic
salts eg.: NaI
presence
2, Adsorption
to silicamatrix
coloumn
2, Washing
+Chelating
agent
+RNase
3, Eluation
with low
salt conc.
solution
DNA
solution
DNA
Denat.
protein
Denaturated protein
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Genomic DNA isolation with magnetic beads
1, Cell lysis:
chaotropic
salts eg.: NaI
in presence
+Chelating
agent
+RNase
2, Adding
silicamatrix
coated
2, Washing
3, Eluation
with low
salt conc.
solution
magnetic
beads
DNA
solution
DNA
Denat. protein
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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.
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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
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Base of RNA isolation
Total RNA specimen
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.
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).
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Purification of plasmid DNA
Denaturated genome-,
plasmid DNA és RNA
1, Cell lysis:
strongly
alkaline
media
Renaturated
plasmid DNA and RNA
3, Alcohol
precipitation
2, Quick
neutralization
of solution pH
3, Washing
Drying
+chelating
agent
Resolving
+ RNase
treatment
Deant. genom
DNA és denat.
protein
+RNA
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Plasmid DNA,
RNA
precipited
+RNA
Plasmid
DNA
solution
4, Determination of nucleicacid solution purity and
concentration
•Based on the absorbance of the solution
Nucleotides and therefore nucleic acids absorb UV radiation.
Absorpiton is maximal at 260nm wavelength and linear at
0-100 µg/ml concentration range
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4, Determination of nucleic acid solution purity and
concentration
1 A260= 40 μg/ml RNS
Absorbance at 260 nm
Absorbance
2,0
240
260
280
300 (nm)
1,5
1,0
0,5
1 A260= 50 μg/ml DNS
20
40
60 80 100 120
Conc.of nucleic acid (μg/ml)
RNA DNA Protein
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Checking nucleic-acid solution purity
A260/A280 > 1.8
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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
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4, Determination of nucleicacid solution purity,
concentration and quality
•Based on gel-electrophoresis
During gel-electrophoresis the electric field moves charged
molecules through the gel. The animating force correlates
with the charge/mass ratio of the molecules while the
retaining force exerted by the gel correlates the size(mass)
of the molecule
Since the charge/mass ratio of nucleic acids is constant
separation of them is based on their size(mass) and shape
and therefore gel-electrophoresis allows determination of
the size of DNA and RNA molecules
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-
Plasmid
Size of DNA molecule :
Based on „band” position
(circular and linear deviates)
Amount of DNA:
Based on „bands” thickness
~1 μg DNA
+
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Linear
Open ring
Linear
Closed ring
(supercoiled)
RNA
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Genomic DNA
Plasmid DNA
+RNA
Plasmid DNA
after adding RNase
Fragmented
chromosomal
DNA
(linear)