DNA evidence is a frozen moment in time Image by collaborator Carl Kriigel, US Army Criminal Investigation Lab DNA is convicting the guilty and freeing the inncocent Clarence Harrison, 2004 Local DNA expert helps overturn Georgia conviction 08:56 AM PDT on Wednesday, September 1, 2004 Adam Atchison / KTVB …Thanks in part to the efforts of Hampikian and his team, Georgia resident Clarence Harrison walked out of the courtroom a free man on Tuesday. Just last week, Hampikian reviewed new DNA test results and discovered Harrison’s DNA doesn't match the evidence saved from the scene of a rape and robbery in 1986 … How good is DNA at exonerating? Crime labs report about 25% of samples sent by law enforcement do not match primary suspect (FBI, GBI, Virginia, Connecticut, Justice Department) Hampikian group mitochondrial projects Other activities: • Basque mitochondrial heritage study of 95 unrelated families • Murder of an Alaskan Native Chief Where Is All This DNA Coming From? • DNA is found in all body cells (except mature red blood cells) • We leave a little bit of DNA everywhere we go • Most forensic sources of DNA are body fluids, or transferred cells • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue DNA Use in Forensic Cases • Most are rape cases (>2 out of 3) • Looking for match between evidence and suspect • Mixtures must be resolved • DNA can be degraded (bacteria, fungi, sunlight, heat) • Inhibitors to diagnostic test can be present (heme, dyes…) • Scientists need a quick and easy way to produce DNA in sufficient quantities for their studies and generate labeled DNA molecules to visualize and study specific molecules within cells. Challenges Modified from www.bioforensics.com PCR (Polymerase Chain Reaction) GV: TS. Lê Quang Nguyên DNA in the Cell Target Region for PCR Target Region for PCR chromosome cell nucleus Double stranded DNA molecule Individual nucleotides www.cstl.nist.gov/biotech/strbase/ppt/4 What is PCR? What is PCR? • PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. • “Polymerase” because the only enzyme used in this reaction is DNA polymerase. • “Chain” because the products of the first reaction become substrates of the following one, and so on. • It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Cetus Corporation, Department of Human Genetics, Emeryville, CA 94608. Development/Invention of PCR Technique 1993 Nobel Prize in Chemistry [...]... thermophilic bacteria discovered in 1969 in hot spring of Yellowstone National park It can tolerate high temperature The DNA polymerase (Taq polymerase) was isolated The PCR Process - PCR Primers • Primers define the DNA sequence to be amplified— they give the PCR specificity • Primers bind (anneal) to the DNA template and act as starting points since DNA polymerases cannot initiate DNA synthesis without a primer... enzyme What is Taq polymerase?     Most proteins denature at extreme pH or high temperatures Human DNA polymerase would denature at 94C New polymerase would have to be added at each elongation step Taq polymerase is the DNA polymerase I for Thermus aquaticus; a bacterium that lives in hot springs Many of its enzymes (including DNAP I) will not denature at high temperatures Thermus aquaticus, a thermophilic... 2004/2/26-195,265 PCR Amplifies a Specific DNA Seq • PCR can be used to target a specific DNA subsequence in a much larger DNA sequence (e.g., a single 1000bp gene from the human genome, which is 3 × 109bp) • PCR allows exponential amplification of a DNA sequence – Each PCR cycle theoretically doubles the amount of DNA – During PCR, an existing DNA molecule is used as a template to synthesize a new DNA strand – Through... allow primers to anneal to their complementary sequence • Primer Extension: Adjust the temperature for optimal thermostable DNA polymerase activity to extend primers PCR uses a thermostable DNA polymerase so that the DNA polymerase is not heat-inactivated during the DNA denaturation step Taq DNA polymerase is the most commonly used DNA polymerase for PCR The PCR Process - Mechanism of DNA Synthesis... The distance between the two primers determines the length of the newly synthesized DNA molecules The PCR Process One PCR cycle consists of a DNA denaturation step, a primer annealing step and a primer extension step • DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers • Primer Annealing: Lower the temperature... repeated rounds of DNA synthesis, large quantities of DNA are produced Advantages of PCR PCR is one of the most useful techniques in laboratories today due to its speed and sensitivity •Traditional techniques to amplify DNA require days or week PCR can be performed in as little as 1-3 hours •Many biochemical analyses require the input of significant amounts and certain purity of biological material;... Synthesis ? Thermal Cycling Programs A typical thermal cycling program is: • Initial DNA denaturation at 95oC for 2 minutes • 20–35 PCR cycles: • Denaturation at 95oC for 30 seconds to 1 minute • Annealing at 42–65oC for 1 minute • Extension at 68–74oC for 1–2 minutes • Final extension at 68–74oC for 5–10 minutes • Soak at 4oC The PCR Process - Instrumentation • Thermal cyclers have a heat-conducting block... • DNA polymerase extends the primer by sequentially adding a single dNTP (dATP, dGTP, dCTP or dTTP) that is complementary to the existing DNA strand • The sequence of the newly synthesized strand is complementary to that of the template strand • The dNTP is added to the 3´ end of the growing DNA strand, so DNA synthesis occurs in the 5´ to 3´ direction The PCR Process - Mechanism of DNA Synthesis... amplify equally well at a number of Mg2+ concentrations, but some reactions only amplify well at a very specific Mg2+ concentration • When first time using a set of PCR primers: titrate magnesium in 0.5 or 1.0mM increments to determine the optimal concentration Primer Annealing Temperature • PCR primers must anneal to the DNA template at the chosen annealing temperature • The optimal annealing temperature... have similar annealing temperatures PCR Primer Design • Ideally all primers used in a PCR will have similar melting temperatures (45–70oC) and GC content (~50%) • Primers should have little intramolecular and intermolecular secondary structure, which can interfere with primer annealing to the template – Primers with intramolecular complementarity can form secondary structure within the same primer . DNA evidence is a frozen moment in time Image by collaborator Carl Kriigel, US Army Criminal Investigation Lab DNA is convicting the guilty and freeing the inncocent Clarence Harrison,. uses a thermostable DNA polymerase so that the DNA polymerase is not heat-inactivated during the DNA denaturation step. Taq DNA polymerase is the most commonly used DNA polymerase for PCR. The. step. • DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers. • Primer Annealing: Lower the temperature