The isolation of inheritance material Cell lysis Isolation of genom DNA Isolation of RNA Isolation of plasmid DNA Determination of nukleic-acid solution

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.



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




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.





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.





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


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

Documents

The isolation of inheritance material Cell lysis Isolation of genom DNA Isolation of RNA Isolation of plasmid DNA Determination of nukleic-acid solution