DNA QUANTIFICATION

forensic genetics

uv spectrophotometry

• base portion of nucleic acids absorb UV light

• quantification of total nucleic acid content

  • no differentiation

• measurements

  • DNA extract diluted into solution (TE buffer or water) 

  • measured against blank (same solution, no DNA extract)

  • absorption blank subtracted from adsorption extract

  • ratio 260:280- estimate of exact purity 

no species specificity- potential influence of microorganism DNA in decomposed samples and overestimation of DNA content

agarose gel electrophoresis + ethidium bromide staining

size and quantification standard- Gene-Ruler

• fragment length band (bp)

ethidium bromide

• interchelating DNA stain

• no species specificity 

evaluation of DNA extracts 

• band- high molecular weight DNA, low/no degradation

• smear- degraded DNA 

• no visible band- DNA concentration below sensitivity limit method

• colour differs from DNA signal- low MW, co-extracted impurity present  

slot blot analysis

• DNA immobilised on nylon membrane

• hybridisation of primate specific DNA probe 

• detection of hybridised probe 

  • radioactive/ fluorescent label attached to probe 

  • chemiluminescent/ colorimetric 

• evaluation quantifying unknown sample

• compared to band intensities standard DNA dilutions of known concentrations 

primate specific probe 

Pico-Green assay

Pico-Green

• interchelating DNA stain, stains dsDNA

• binding to dsDNA- increase in fluorescence 

no species specificity 

staining of DNA and fluorescence detection in solution (480nm,520nm)

quantification against standard curve of known concentrations 

PCR

thermal cycling steps 

• denaturation

  • 90-95oc

  • DNA strands separate 

• annealing

  • 50-60oc

  • primers bind complementary to DNA target 

• elongation 

  • 72oc

  • DNA polymerase extends primer across target sequence using DNTPs 

exponential growth → linear growth → plateau

PCR 

reaction components 

• DNA polymerase

  • strand elongation (polymerisation)

• MgCl2

  • co-factor/ co-enzyme necessary for activity DNA polymerase 

• KCl (or (NH4)2SO4)

  • supports primer annealing and stability in primer-template complex

• water (purified, DNA free)

  • aquatic system 

  • used to bring reaction mix to required volume 

• primers (2 per locus)

  • synthetic digonucleotides 

  • compliment to flanking region locus of interest 

  • start points for synthesis complementary DNA strand

• DNTPs

  • nucleic monomers for elongation of complementary DNA strand

• tris-HCl (ph 8- 9.2)

  • enzyme activity is dependent on ph

taq polymerase - ph 9

quantitative PCR

PCR amplification target sequence- increase fluorescence signal

real time analysis, monitoring cycle to cycle changes 

quantitative PCR

5’ nuclease assay (TaqMan assay)

• dual labelled probe

  • reporter dye + quencher dye

• probe hybridises to sequence within target region

• target amplification: DNA polymerase cleaves probe 

  • ← 5’ nuclease activity

• separation quencher dye from reporter dye

  • reporter dye emits fluorescence 

• detection target specific probe

• target specific DNA quantification 

locus specific

quantitative PCR

SYBR Green assay

SYBR Green

• interchelating dye- binds to dsDNA

• emits fluorescence only when bound to dsDNA

detection of dsDNA specific dye

• non target specific DNA quantification

• general dsDNA specific DNA quantification 

requires melting curve analysis

real time quantitative PCR

(graph with all the PCR lines with cycle number)

• cT- threshold cycle

  • cycle at which curve exceeds threshold

• cT plot against Log[DNA]

  • use graph to find unknown

Nc = No (1 + E)^c

• if efficiency is close to 100% (E=1) then the product copy number (Nc) doubles the target copy number (No) with each cycle number (c )

quantitative PCR

end point analysis- 

1) PCR amplification

• unknown sample to be quantified 

• dilution series of standard DNA sample with known concentration 

2) detection of PCR products

• e.g. gel electrophoresis + ds staining 

• e.g. pico green assay + fluorometer/plate reader

3) quantification of unknown sample

• comparison PCR product yield unknown sample to dilution series unknown sample

quantitative PCR

applications

quantification of DNA, more specific amplifiable DNA

amplification of DNA 

• more specific defined portion of DNA = target loci

• selective amplification of specific portions of DNA = target locus

• no DNA xeroxing

  • not 1:1 copy but small portion is copied