GENOMIC DNA: ISOLATION AND APPLICATIONS

Cell and Molecular Biology Lab Department of Biological Sciences

UST College of SciencePost Lab Discussion # 6

Genomic DNA Sum total of all DNA inside the

nucleus Nuclear DNA Chromosomal DNA Genome – the haploid set of

DNA (3 x 109 bp) Different from:

Mitochondrial DNA Chloroplast DNA Extrachromosomal DNA (plasmids)

Classification of Genomic DNA

• Protein- coding genes– Solitary– Duplicated/ diverged

• Tandemly repeated genes

• Repetitive DNA– Simple sequence– Moderately repeated– Mobile elements

• Spacer DNA

GENOMIC DNA ISOLATION FROM HUMAN BLOOD

Separation of RBC and WBC RBC lysis

10 mM Tris-Cl 0.15 mM NH4Cl/ 10 mM KHCO3/ 5% EDTA MgCl2- Slightly acidic solutions

Blood fractionation Cell lysis

Tris-Cl Sodium salt SDS

Protein Precipitation Potassium Acetate

DNA Precipitation Absolute alcohol

Washing 70% alcohol

DNA renaturation Tris-EDTA buffer

GENOMIC DNA ISOLATION FROM RICE SEEDLINGS• DNA extraction buffer

– Cell wall and cell membranes’ disruption

• Protein denaturation– Chloroform/ phenol

• DNA precipitation– Absolute alcohol

• Washing– 70% alcohol

• DNA renaturation– TE buffer

Assessing the Integrity of DNA High Quality Genomic DNA

>95% DNA will be of high molecular weight, migrating as intact band near the top of the gel

Very little evidence of smaller fragments indicated by a smear of many different sized DNA fragments

DNAA260 1.0 50 ug/mL

A260 / A280 1.6-1.8 High Purity

RNA

A2601.0 (in water)

40 ug/mL

A260 / A280

1.9-2.1 (in 10 mM Tris-Cl, pH 7.5)

High Purity

Genomic DNA from 8 blood samples stored at 4°C for 1 week. DNA was purified using the QIAamp DNA Blood Mini Kit. When blood is stored at 4°C the DNA is rapidly degraded due to apoptosis; the resulting apoptotic banding pattern can clearly be seen in these samples. M1: lambda–HindIII; M2:100 bp ladder.

Agarose gel electrophoresis of DNA purified with SpinClean™ Genomic DNA purification Kit.M : 1 kb ladder marker, line 1: Chicken whole blood(20ul sample volume), line 2 : Human whole blood(100ul sample volume), line 3 : E.coli, line 4 : L.brevis, line 5 : Streptomyces hygroscopicus subsp.

INTACT DNA SAMPLES DEGRADED DNA SAMPLES

4 bio 2

4 bio 3

4 bio 4

4 bio 5

4 bio 6

USES OF ISOLATED GENOMIC DNA

• Preparation of genomic libraries• PCR template• Cloning• Gene/DNA sequencing• Analysis of genomic organization• Study gene structure• DNA fingerprinting• Analysis of genome composition• Detection of abnormalities / mutations

Polymerase Chain Reaction (PCR)

Rapid procedure for in vitro enzymatic amplification of a specific segment of DNA

Developed by Kary Mullis in 1985 (Nobel prize in 1993)

Theoretical basis was first described by Kleppe K. in 1971

Based on the principle that double-stranded (ds) DNA denatures at high temperatures and DNA polymerase synthesizes ds DNA in the presence of template and primer

Exponential increase of ds DNA produced with increasing cycles

PCR Machine (Thermal Cycler)

Automated machine that controls the changes in temperature needed in different steps in PCR: Denaturation temperature

Annealing temperature

Extension temperature

PCR Mixture

Template DNA - 1.0 uL

Forward Primer - 1.0 uL

Reverse Primer - 1.0 uL

dNTPs - 1.0 uL

PCR Buffer - 2.0 uL

Enzyme - 0.4 uL

ddwater - 13.6 uL

TOTAL - 20.0 uL

PCR STEPS

Template DNA Quality of DNA template is important

Salts, guanidine, proteases organic solvents and SDS affect PCR

Verify purity of sample by electrophoresis or spectrophotometry

Ethanol precipitation removes most contaminants

Amount of template required: <10 ng/uL of DNA

Different DNA samples have different amounts of the target DNA

Example: 4 kb plasmid with 1 kb insert (25% of the total DNA is the target DNA)

1 kb gene in human genome = 0.00003% (3.3x109)

Primer Design 15-30 mers (bases)

40-60 % GC content

Avoid sequences forming secondary structure

3’ ends should NOT be complementary to avoid primer dimers

Avoid 3 G or C in a row near the 3’ end

Both primers should anneal at the same temperature

Annealing temp is dependent on the primer with lower melting temp

Final concentarion should not exceed 50pmol (1 uM concentartion)

Melting Temperatures of Primers

Tm = (81.5 + 16.6)(log10[Na+])+(0.41)(%G+C)-675/n

Where [Na+] = molar salt concentration

n = number of bases in oligonucleotides

Tm = [(A+T)2] + [(C+G)4]

ATG GCG GCT CGA TCA GCA AA

Tm = [(9)2]+[(11)4] 62

Enzyme Concentration Recommended: 1.25 U of Taq pol in a 50 uL

reaction( elongation: 100-500 bp/min)

Inclusion of more enzyme does not significantly increase product yield

High amounts of enzymes results to artifacts in PCR products

Prepare master mixes to avoid pipeting errors (enzymes are stored in glycerol – accurate pipeting is almost impossible)

Enzyme Concentration Recommended: 1.25 U of Taq pol in a 50 uL

reaction

Inclusion of more enzyme does not significantly increase product yield

High amounts of enzymes results to artifacts in PCR products

Prepare master mixes to avoid pipeting errors (enzymes are stored in glycerol – accurate pipeting is almost impossible)

Magnesium Concentration Affects the performance of polymerase

Taq polymerase is inactive in the absence of free Mg

Excess free Mg reduces enzyme fidelity and may increase non-specific amplification

Determine optimal Mg concentration (1-3 mM)

Use 0.5 mM increments

Completely thaw MgCl before use

Magnesium chloride could form precipitates in frozen state – vortex before use

Amplification It has an exponential increase in

the number of target DNA fragments

30 cycles = 230 copies

Y= A x 2n

A= initial number of copies of target DNA

N= number of cycles

Y number of copies of DNA after the reaction

Time

Amount of DNA

Applications of PCR Direct cloning from genomic DNA or cDNA

In vitro mutagenesis and genetic engineering

Genetic fingerprinting of forensic samples

Assays for the presence of infectious agents

Prenatal diagnosis of genetic diseases

Analysis of allelic sequence variations

Analysis of RNA transcript structures

Genomic footprinting

Direct nucleotide sequencing of genomic and cDNA

DNA FINGERPRINTING

DNA FINGERPRINTING• RFLPs- Restriction Fragment

Length Polymorphisms• VNTRs – Variable Number

Tandem Repeats

Crime Investigation

Paternity testing

Can you tell who among the children are real sons/ daughters? Adopted? Step-daughter/son?