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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

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Route of gemon DNA isolation from tissues to
applications
White blood cells
Plant, aninal tissues
Cultivated cells
Embrional cells
Forensic samples
Fossils
Genebank Molecular PCR Sequencing
Southern-blot Examination of polymorphism
cloning
SNP,VNTR, RFLP
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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
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Tissue sample/cell isolation and cell lysis,
making of DNA solution

• 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
Plant, animal tissues enzymatic cell wall
digest, Homogenizazor with knifes, frozen
grinding
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Tissue sample/cell isolation and cell lysis,
making of DNA solution

• 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
Cultivated cells disattachment trypsin
digestion Cell lysis hipotonic shock, detergent
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Tissue sample/cell isolation and cell lysis,
making of DNA solution
White blood cells hypotonic shock, detergent
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.
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.
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Tissue sample/cell isolation and cell lysis,
making of DNA solution

• 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

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
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Tissue sample/cell isolation and cell lysis,
making of DNA solution
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.
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.
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Route of gemon DNA isolation from tissues to
applications
White blood cells
Plant, aninal tissues
Cultivated cells
Embrional cells
Forensic samples
Fossils
Genebank Molecular PCR
Sequencing Southern-blot
Examination of polymorphism cloning

SNP,VNTR, RFLP
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(No Transcript)
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Genomic DNA isolation with pronase treatment,
phenol extraction and precipitation with alcohol
Pronase treatment
Phenol extraction
Alcohol precipitation
Cell lysis Chelation
DNA RNA
denat. proteins
phenol
DNA RNA precipitate
DNA RNA Protein
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
Cell lysis chaotropic salts eg.
NaI presence Chelating agent RNase
Sample application on silica- matrix coloumn
Washing
Eluation with low salt conc. solution
DNA solution
DNA Denat. protein
Denaturated protein
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Genomic DNA isolation with magnetic beads
Adding silica- matrix coated magnetic beads
Eluation with low salt conc. solution
Cell lysis chaotropic salts eg. NaI in
presence Chelating agent RNase
washing
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 BioP
rotocol http//www.bio.com/protocolstools/discipl
ine.jhtml?idpc1
Genomic DNA isolation on affinity coloumn
http//www.genomed-dna.com/G_M03_03.htm http//ww
w.clontech.com/clontech/techinfo/faqs/mn.shtml ht
tp//www1.qiagen.com/
Genomic DNA isolation with magnetic
beads GenoPrep Cartridge www.genovision.com
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Base of RNA isolation
The RNA isolation is based on similar principle
as the genomic DNA separation Characteristics RN
ase 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/t
echlib/basics/rnaisol/ index.html Qiagen http//ww
w1.qiagen.com/ Downstreem applications Northern
analysis, RT-PCR, in vitro translation,
expression profile determination (DNA chips)
and cDNA library construction.
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
Renaturated plasmid DNA and RNA
Denaturated genome-, plasmid DNA és RNA
Cell lysis strongly alkaline media chelating
agent
Alcoholic precipitated
Washing Drying Resolving RNase treatment
Quick neutralization of solution pH
Deant. genom DNA és denat. protein
Plasmid DNA, RNA precipited
Plasmid DNA solution
RNA
RNA
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Determination of nucleic-acid solution purity
and concentration

• Based on absorbency value of solution
• Based on gelelectrophoresis image

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RNA DNA Protein
Absorbency
240 260 280
300 (nm)
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Checking nucleic-acid solution purity
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2,0 1,5 1,0 0,5
RNA DNA
Absorbancy at 260 nm
20 40 60 80
100 120
Nukleinacid concentration (µg/ml)
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Checking the purity of nucleic-acid solution
A260/A280 gt 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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Determination of nucleic-acid solution purity
and concentration

• Based on value of solution absorbency
• Based on gelelectrophoresis image

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Plasmid Linear
Size of DNA molecule Based on band
position (circular and linear deviates) DNS
amount Based on bands thickness
1 µg DNA

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• Practical
• Genomic DNA isolation from homogenized pig liver
  cells
• pUC19 (2686 bp) plasmid isolation Escherichia
  coli DH5a
• from a laboratorial bacterial strains
• Determination of nukleicacid solution purity
• and concentration with gelelektroforezis

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Plasmid DNA after adding RNase
Plasmid DNA RNA
Genomic DNA
Fragmented chromosomal DNA (linear)
Open ring
Linear
Closed ring (supercoiled)
RNA
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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