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3rd lecture PCR-Presentation.ppt

G
gayubshah

The document discusses the polymerase chain reaction (PCR) technique. It describes how PCR allows targeted amplification of specific DNA regions. Key steps in PCR include using DNA polymerase, primers, and thermal cycling to denature and replicate the DNA. The document outlines different types of PCR like quantitative, multiplex, and nested PCR. It also discusses how PCR has revolutionized fields like molecular archaeology by enabling the cloning of ancient DNA. PCR is now widely used in applications like forensics, disease diagnosis, and genome sequencing.

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Introduction
• PCR, polymerase chain reaction, is an in-vitro
technique for amplification of a region of DNA
whose sequence is known or which lies
between two regions of known sequence
• Before PCR, DNA of interest could only be
amplified by over-expression in cells and this
with limited yield
• 1966, Thomas Brock discovers Thermus
Aquaticus, a thermostable bacteria in the
hot springs of Yellowstone National Park
• 1983, Kary Mullis postulated the concept of
PCR ( Nobel Prize in 1993)
• 1985, Saiki publishes the first application of
PCR ( beta-Globin)
• 1985, Cetus Corp. Scientists isolate
Thermostable Taq Polymerase (from
T.Aquaticus), which revolutionized PCR
Reaction Components
• DNA template
• Primers
• Enzyme
• dNTPs
• Mg2+
• buffers
1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb
2- Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to the
3’ ends of target DNA
• not complimentary to
each other
Primers (ctnd)
• Not containing inverted repeat sequences to
avoid formation of internal structures
• 40-60% GC content preferred for better
annealing
• Tm of primers can be calculated to determine
annealing T0
• Tm= .41(%G+C) + 16.6log(J+) + 81.5 where J+ is
the concentration of monovalent ions

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3rd lecture PCR-Presentation.ppt

  • 1. Introduction • PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence • Before PCR, DNA of interest could only be amplified by over-expression in cells and this with limited yield
  • 2. • 1966, Thomas Brock discovers Thermus Aquaticus, a thermostable bacteria in the hot springs of Yellowstone National Park • 1983, Kary Mullis postulated the concept of PCR ( Nobel Prize in 1993) • 1985, Saiki publishes the first application of PCR ( beta-Globin) • 1985, Cetus Corp. Scientists isolate Thermostable Taq Polymerase (from T.Aquaticus), which revolutionized PCR
  • 3. Reaction Components • DNA template • Primers • Enzyme • dNTPs • Mg2+ • buffers
  • 4. 1- DNA template • DNA containing region to be sequenced • Size of target DNA to be amplified : up to 3 Kb
  • 5. 2- Primers • 2 sets of primers • Generally 20-30 nucleotides long • Synthetically produced • complimentary to the 3’ ends of target DNA • not complimentary to each other
  • 6. Primers (ctnd) • Not containing inverted repeat sequences to avoid formation of internal structures • 40-60% GC content preferred for better annealing • Tm of primers can be calculated to determine annealing T0 • Tm= .41(%G+C) + 16.6log(J+) + 81.5 where J+ is the concentration of monovalent ions
  • 7. 3-Enzyme • Usually Taq Polymerase or anyone of the natural or Recombinant thermostable polymerases • Stable at T0 up to 950 C • High processivity • Taq Pol has 5’-3’ exo only, no proofreading
  • 8. The PCR Cycle • Comprised of 3 steps: - Denaturation of DNA at 950C - Primer hybridization ( annealing) at 40-500C - DNA synthesis ( Primer extension) at 720C
  • 15. RT-PCR • Reverse Transcriptase PCR • Uses RNA as the initial template • RNA-directed DNA polymerase (rTh) • Yields ds cDNA
  • 19. Detection of amplification products • Gel electrophoresis • Sequencing of amplified fragment • Southern blot • etc...
  • 23. Types of PCR: 1) Quantitative PCR techniques It is also referred to real time PCR. It gives an idea about how much DNA amount present in the sample. 2) Qualitative PCR techniques When PCR techniques is used for detecting a specific DNA segment, it is called as quantitative PCR method. PCR techniques are used in the identification of genes of bacteria and virus (9). Only qualitative PCR technique can detect whether the individual has been reinfected with a different but related pathogen. It is fast and simple, inexpensive technique.
  • 24. 3) Conventional PCR This defined as a normal PCR process. Here the primers bind specifically to each other with 2 DNA strands. Primers also limit the sequence to be replicated and a particular DNA sequence is amplified with billions of copies. All that needed for the PCR process are PCR tubes made up of aluminium blocks, DNA polymerase, buffer, and target DNA, primers. The whole process takes place within 35-40 minutes repeatedly and viewed by gel electrophoresis technique.
  • 25. 4) Multiplex PCR • Multiplex Pcr technique detects different pathogens in a single sample, used to identify exonic/intronic sequence5 in specific genes. • The designing of primers are different because they are meant to adhere to specific DNA sequence. • Here in multiplex PCR the base pair lengths should be different to form distinct bands because varying sizes of different DNA genes are targeted in a single reaction to avoid higher expenses, time consumption and recognizes many pathogens at once11. • This technique is used to detect viral/bacterial and other infectious agents. • The presence of one or more primer pair increases the risk of primer-dimer amplification and discrimination of DNA laer fragments10.
  • 26. 5) Nested-semi nested PCR • Two sets of primers are used here for a single locus point. • The first set is an amplified sequence and the second set is complementary to the first sequence which will be shorter than the first amplified product. • Nested PCR is used because it intends to reduce the contaminations in products due to the amplification of unexpected primer binding sites. • It has drawbacks like risk of contamination and needs great care while being performed. • These contaminations can be controlled by adding ultra-pure oil of two mixtures,5 and by using primers designed to anneal at different temperatures. • This method is more specific but has disdvantages like ‘primer and dimerisation’17 cross reaction
  • 27. 6) Quantitative/semiquantitative-real time PCR Fluorescent dyes like SYBER Green master mix are used for the identification of samples and probes are used to measure the amount of amplified product in real- time2. cDNA is obtained by RT-PCR for a RNA sample. ApoA1/Bactin are used as markers followed by gel electrophoresis process with ethidium bromide dye staining procedure. Here the main disadvantage is the generation of non-specific hybridisation. All the reactions were performed in quadruplicates using the DNA stocks.
  • 29. This technology is highly sensitive: • PCR can detect and amplify as little as one DNA molecule in almost any type of sample. • Although DNA degrades over time PCR has allowed successful cloning of DNA from samples more than 40,000 years old. • Investigators have used the technique to clone DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth, creating the new fields of molecular archaeology and molecular paleontology. • DNA from burial sites has been amplified by PCR and used to trace ancient human migrations. • Epidemiologists can use PCR-enhanced DNA samples from human remains to trace the evolution of human pathogenic viruses. • PCR is a potent tool in forensic medicine. • It is also being used for detection of viral infections before they cause symptoms and for prenatal diagnosis of a wide array of genetic diseases. • The PCR method is also important in advancing the goal of whole genome sequencing.
  • 30. Applications • Genome mapping and gene function determination • Biodiversity studies ( e.g. evolution studies) • Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections...) • Detection of drug resistance genes • Forensic (DNA fingerprinting)
  • 31. Advantages • Automated, fast, reliable (reproducible) results • Contained :(less chances of contamination) • High output • Sensitive • Broad uses • Defined, easy to follow protocols