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Polymerase Chain Reaction (PCR)

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Dr. Sumbul Fatma Department of Medical Biochemistry What is PCR? It s a means of selectively amplifying a particular segment of DNA Each cycle of amplification ... – PowerPoint PPT presentation

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Title: Polymerase Chain Reaction (PCR)


1
Polymerase Chain Reaction (PCR)
  • Dr. Sumbul Fatma
  • Department of Medical Biochemistry

2
What is PCR?
  • Its a means of selectively amplifying a
    particular segment of DNA
  • Each cycle of amplification doubles the amount of
    DNA in the sample
  • Source of DNA could be any- bacterial, viral,
    plant and animal

3
Advantages of PCR?
  • PCR allows the DNA in a single cell, hair
    follicle, or spermatozoan to be amplified and
    analyzed
  • DNA sequences as short as 50-100bp and as long as
    10kb can be amplified
  • As few as 20 cycles would yield 106 times the
    amount of target DNA initially present

4
The invention of PCR
  • Invented by Kary B Mullis in 1983
  • First published account appeared in 1985
  • Awarded Nobel Prize for Chemistry in 1993

5
Requirements of a PCR
  • DNA polymerase- to repetitively amplify targeted
    portion of DNA
  • Nucleotide triphosphates- ATP, GTP, CTP and TTP
  • Primers- two single stranded oligonucleotides
    (20-25ntds long), which are complimentary to the
    flanking sequences that bracket the target DNA
    sequence

6
Requirements of a PCR
  • Thermal Cycler
  • PCR cyclers are available from many suppliers
  • Reactions are done in tubes or 96 well microtitre
    plates

7
Steps of a PCR
  • Primer construction- it is synthetic
    oligonucleotide complimentary to the short
    nucleotide segments on each side of the target DNA

8
Steps of a PCR
  • Denature the DNA- The DNA to be amplified is
    heated to separate the double stranded target DNA
    into single strands(1 min. 940C )

9
Steps of a PCR
  • Annealing of primers to ssDNA- the separated
    strands are cooled and allowed to anneal to the
    two primers (one for each strand)

45 sec, 540C Forward and reverse primers
10
Steps of a PCR
  • Chain extension- the DNA polymerase adds
    nucleotides to the 3-hydroxyl end of the primer,
    and strand growth extends across the target DNA,
    making complimentary copies of the target (2 min
    720C)
  • At the completion of one cycle of replication,
    the reaction mixture is heated again to denature
    the DNA strand (of which there are now 4)

11
Polymerase Chain Reaction
12
Thermocycling
  • Denaturation - 940C
  • Annealing - 550C
  • Extension - 720C
  • Denaturation again.
  • 20-30 cycles
  • The amplified target sequence is called amplicons
  • With each cycle there is an exponential increase
    in the amount of target DNA, hence the name
    Polymerase Chain Reaction

13
DNA polymerase in PCR
Heat stable DNA polymerase is vital to the ease
of the process
14
Taq DNA polymerase in PCR
Thermus aquaticus, a thermophilic bacteria that
lives and replicates at 70-800C is the source of
Taq DNA polymerase used in PCR reactions.
15
Multiple cycles of PCR
16
The target is RNA ?
  • the RNA must be enzymatically converted to DNA
  • Reverse Transcriptase- are the RNA directed DNA
    polymerases
  • Reverse Transcriptase
  • RNA cDNA
  • cDNA is then amplified by PCR
  • This process is termed as RT-PCR

17
Analysis of PCR products
  • Amplicons can be analyzed by gel electrophoresis
    and Southern Blot- qualitative analysis
  • Quantitative PCR- used to measure the viral loads
    in HIV and Hep C-infected patients
  • These numbers allow physicians to determine
    disease status and evaluate efficacy of antiviral
    treatment.

18
Real Time PCR
  • Does not measure the amount of end product of PCR
    but its production or accumulation in real time
  • Two common methods of quantification are-
  • 1. the use of fluorescent dyes that
    intercalate with double-stranded DNA e.g. SYBR
    green
  • 2. modified DNA oligonucleotide probes that
    fluoresce when hybridized with a complementary
    DNA (Taqman probes, molecular beacons and
    scorpion primers)

19
Real Time PCR- detection methods
  • Fluorescent dyes like SYBR green-
  • A DNA-binding dye binds to all dsDNA in PCR,
    causing fluorescence of the dye. An increase in
    DNA product during PCR therefore leads to an
    increase in fluorescence intensity and is
    measured at each cycle, thus allowing DNA
    concentrations to be quantified

20
Real Time PCR- detection methods
  • Unhybridized probe has donor fluorophore and
    non-fluorophore acceptor molecule (quenchers) in
    close proximity no signal
  • Upon hybridization to the target, the
    fluorophore and quencher become separated through
    either
  • Conformational change- molecular beacons,
    scorpion primers
  • Enzymatic cleavage of the fluorophore from the
    quencher as a result of 5 to 3 nuclease
    activity of the Taq DNA polymerase- Taqman probes

21
Applications of PCR
  • Comparison of a normal cloned gene with an
    uncloned mutant form of the gene
  • Detection of low abundance nucleic acid sequences
    e.g. viruses, mRNA in cells or tissue
  • Forensic analysis of DNA sample
  • Prenatal diagnosis and carrier detection of
    Cystic Fibrosis

22
Cystic Fibrosis
  • It is an autosomal recessive disorder
  • Results from mutations in the cystic fibrosis
    transmembrane conductance regulator gene
  • The most common mutation is loss of Phe residue
    from the protein
  • Distinguished by difference in size of the
    mutated PCR product

23
References
  • Lippincott s Illustrated Reviews, 4th Edition
  • Clinical Chemistry Principles, Procedures,
    Correlations by Michael L Bishop
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