PCR

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PCR

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PCR (Polemerase Chain Reaction, phản ứng chuỗi polymerase) là một kỹ thuật nhằm tạo ra một lượng lớn bản sao DNA mục tiêu trong ống nghiệm dựa vào các chu kỳ nhiệt. Kỹ thuật này được nhà khoa học người Mỹ Kary Mullis phát minh vào năm 1985.

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  1. DNA evidence is a frozen moment in time Image by collaborator Carl Kriigel, US Army Criminal  Investigation Lab
  2. 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 …
  3. 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)
  4. Hampikian group mitochondrial projects Other activities: • Basque mitochondrial heritage study of 95 unrelated families • Murder of an Alaskan Native Chief
  5. 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 • Saliv a • Urine • Hair • Teeth • Bone • Tissu
  6. DNA Use in Forensic Cases • Most are rape cases (>2 out of 3) • Looking for match between evidence and suspect Challenges • 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. Modified from
  7. PCR (Polymerase Chain Reaction) GV: TS. Lê Quang Nguyên
  8. DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides www.cstl.nist.gov/biotech/strbase/ppt/4
  9. 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.
  10. Development/Invention of PCR Technique 1993 Nobel Prize in Chemistry 
  11. PCR: Polymerase Chain Reaction • A method of in vitro cloning • Allows amplification of specific DNA molecules (fragments) in vitro through cycles of enzymatic DNA synthesis • The most popular and widely used technique in all fields of biological studies probably. • 1989 - 219 • 1990 – 496 1998/10 - >73,000 • 1991 – 711 1999/4 - >81,000 • 1992 – 906 2000/10 – 121,305 • 1993 –1030 2001/2 – 125,563 • 1994 – 857 2002/3 – 149,572 • 1995 – 823 2003/2 – 170,841 • 1996 – 796 2004/2/23-195,193 • 1997 – 732 2004/2/26-195,265 • 2006/3/22 - 255,788 • 2006/4/18 – 257,737 • 2007/3/9 – 283,607 • 2007/4/11 - 286,486
  12. 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 repeated rounds of DNA synthesis, large quantities of DNA are produced.
  13. 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; PCR requires as little as one DNA molecule. •Robustness: will even work on degraded DNA or fixed DNA •Simplicity: require eppendorf, thermocycler, and ingredients for PCR •These features make PCR extremely useful in basic research and commercial applications, including genetic identity testing, forensics, industrial quality control and quick in vitro diagnostics (virus, bacteria,etc.).
  14. Disadvantages of PCR • Need for Target DNA sequence information – To construct primers you need to know your target • Short size limit for product – There is an upper limit to the size of DNA synthesized by PCR • Infidelity of replication – Because the PCR polymerases are heat stable they tend not to have the 3’->5’ exonuclease activity. (Taq  polymerase generates 1 error in 10000 nucleotides) • Impurity
  15. The “Reaction” Components arget DNA ­ contains the sequence to be amplified. r of Primers ­ oligonucleotides that define the sequ amplified. NTPs ­ deoxynucleotidetriphosphates: DNA building bl 4) Thermostable DNA Polymerase ­ enzyme that  catalyzes the reaction 6) Buffer solution – maintains pH and ionic  strength of the reaction solution suitable for  the activity of the enzyme 
  16. 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 in hot spring ofwillbacteria DNAP I) Yellowstone Thermus  aquaticus,  a thermophilic discovered in 1969 not denature at high temperatures It can tolerate high temperature. National park  . The DNA polymerase (Taq polymerase) was isolated.
  17. 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. • The distance between the two primers determines the length of the newly synthesized DNA molecules.
  18. 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 to 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.
  19. The PCR Process - Mechanism of DNA Synthesis • 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.
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