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