Download 01. PCR and QPCR2

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 doubles the amount of
DNA in the sample
 Source of DNA could be any- bacterial, viral, plant
and animal
Dr. Sumbul Fatma
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
Dr. Sumbul Fatma
The invention of PCR
 Invented by Kary B Mullis
in 1983
 First published account
appeared in 1985
 Awarded Nobel Prize for
Chemistry in 1993
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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
Dr. Sumbul Fatma
Requirements of a PCR
Thermal Cycler
 PCR cyclers are available
from many suppliers
 Reactions are done in tubes
or 96 well microtitre plates
Dr. Sumbul Fatma
Steps of a PCR
 Primer construction- it is synthetic oligonucleotide
complimentary to the short nucleotide segments on
each side of the target DNA
Dr. Sumbul Fatma
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 )
Dr. Sumbul Fatma
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
Dr. Sumbul Fatma
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)
Dr. Sumbul Fatma
Polymerase Chain Reaction
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”
Dr. Sumbul Fatma
DNA polymerase in PCR
Heat stable DNA
polymerase is vital to
the ease of the
process ……
Dr. Sumbul Fatma
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.
Dr. Sumbul Fatma
Multiple cycles
of PCR
Dr. Sumbul Fatma
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
Dr. Sumbul Fatma
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.
Dr. Sumbul Fatma
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 are1. 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)
Dr. Sumbul Fatma
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
Dr. Sumbul Fatma
Real Time PCR- detection methods
Unhybridized probe has donor fluorophore and nonfluorophore 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
Dr. Sumbul Fatma
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
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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
Dr. Sumbul Fatma
References
 Lippincott ‘s Illustrated Reviews, 4th Edition
 Clinical
Chemistry:
Principles,
Correlations by Michael L Bishop
Procedures,
Dr. Sumbul Fatma