Genomic Dna Isolation And Applications

GENOMIC DNA: ISOLATION AND APPLICATIONS Cell and Molecular Biology Lab Department of Biological Sciences UST College of Science Post 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

DNA

A260

1.0

50 ug/mL

A260 / A280

1.6-1.8

High Purity

A260

1.0

RNA A260 / A280

(in water)

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

40 ug/mL High Purity

INTACT DNA SAMPLES

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.

DEGRADED DNA SAMPLES

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.

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

Amount of DNA

 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

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? Stepdaughter/son?