qPCR in Forensics

qPCR in Forensics - Steps in Forensic DNA analysis 1...

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Real-Time or Quantitative PCR (qPCR) in Forensics Presented by John E. Schienman, Ph.D. DNA Advisory Board (DAB) Guideline 9.4.2.1 Quantitation Standards that estimate the amount of human nuclear DNA recovered by extraction Steps in Forensic DNA analysis 1) Extract DNA from biological stain 2) Evaluate DNA quality and approximate quantity (agarose yield gel) 3) Quantitate the amount of Human DNA in extract 4) Amplify DNA using STR genotyping kit/s 5) Separate and Detect amplified DNA on 3130 Genetic Analyzer 6) Determine DNA profile from 3130 output with software STR Genotyping kits require a specific range of input DNA for optimal results (0.15 ng – 1.5 ng). Too little, no DNA profile Too much, high background and artifact peaks make mixture interpretation difficult Quantitation methods prior qPCR DNA Hybridization without amplification e.g. Quantiblot and Aluquant qPCR is more sensitive, greater dynamic range, uses less sample, and is less labor intensive for analyst. But, initial cost is high…Real-Time PCR machine costs ~ $75,000 DNA in the Cell Target Region for PCR Target Region for PCR chromosome cell nucleus Double stranded DNA molecule Individual nucleotides DNA Polymerase + Thermostable DNA polymerase, dNTPs, MgCl 2 Many-fold excess of oligo-primers specific for target 5’-GTTGTTCCAGTCATCCCT-3’OH OH3’-AACACCTGCCATGAAGAC-5’ Denature (95°C) & Anneal (50°-65°C) GTTGTTCCAGTCATCCCT 5’ TTGTGGACGGTACTTCTG 3’ CAACAAGGTCAGTAGGGA AACACCTGCCATGAAGAC 3’ 5’ 3’ 3’ CAACAAGGTCAGTAGGGA 5’-GTTGTTCCAGTCATCCCT TTGTGGACGGTACTTCTG AACACCTGCCATGAAGAC 5’ TTGTGGACGGTACTTCTG AACACCTGCCATGAAGAC-5’ GTTGTTCCAGTCATCCCT CAACAAGGTCAGTAGGGA 5’ Extension (60°-72°C) New strands Original strand Original strand 5’-GTTGTTCCAGTCATCCCT-3’OH Primer 1 OH3’-AACACCTGCCATGAAGAC-5’ Primer 2 Repeat Denature & Anneal 5’ 3’ 5’ 3’ TTGTGGACGGTACTTCTG AACACCTGCCATGAAGAC Template containing target for amplification GTTGTTCCAGTCATCCCT CAACAAGGTCAGTAGGGA Target segment of DNA 5’-GTTGTTCCAGTCATCCCT TTGTGGACGGTACTTCTG AACACCTGCCATGAAGAC-5’ CAACAAGGTCAGTAGGGA New strand New strand Make copies ( extend primers ) Starting DNA Template 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ Add primers ( anneal ) 5’ 3’ 3’ 5’ Forward primer Reverse primer DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands ( denature ) 5’ 5’ 3’ 3’
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In 30 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created In 30 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle Some nice animated cartoons at Promega.com navigate thru products, then genetic identity What is Real-Time or Quantitative PCR?
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