M.

Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

PCR methods
PCR amplifies a given sequence in an exponential
fashion

In a perfect world, after 35 cycles, one starting
copy of a gene would yield 2
36
copies of that DNA
fragment68 billion copies

Starting with 100 copies would yield 3.73 ug of
DNA, plenty to be able to sequence, clone and
visualize on an agarose gel



difficult to sequence one copy of a gene and
detect

very easy to sequence some of the 68 billion
copies and detect

PCR
Steps in PCR reaction


denaturation
separate parent strands in
preparation new strand
synthesis
PCR


annealing
stick primers to the
parent strands to prime
DNA synthesis

DNA synthesis requires a
primer to start DNA
synthesis
PCR

extension
addition of nucleotides,
one at a time, to the
growing end of the DNA
strand (3 end) using the
parent strand as the
template

cycle through 25-35
times

PCR animation
PCR
Considerations
incorrect PCR product size (primer design)
mis-priming
too low annealing temperature
redundant sequences

no PCR product
no priming
too high annealing temperature
primer dimers
complementary sequence in primers and theyll
anneal
too much template
forgot enzyme
forgot dNTPs
too much/too little MgCl
2


PCR product with errors
wrong temperature
annealing
extension
too much/too little MgCl
2

affects polymerase proof reading
complex sequence
GGG repeats
low fidelity enzyme
Taq polymerase vs Pfu


RT-PCR reverse transcriptase-pcr
RNA containing virus
PCR doesnt work on
RNA templates

RT PCR
make cDNA copy of RNA
sequence first

PCR the cDNA copy of
RNA
Extract RNA from virus/cells
RNA
RT-PCR
Comparison of Real Time vs traditional PCR
http://www.appliedbiosystems.com/support/tutorials/pdf/rtp
cr_vs_tradpcr.pdf


RT detection methods
Fluorescence quenching
the fluorescence of a fluorescent
molecule can be quenched by
close proximity to a quenching
agent
upon removal of the quencher,
the fluorescence siganl returns

Exonuclease activity
DNA polymerase
ability to synthesize new DNA
strand
ability to remove nucleotides
from a second strand of DNA as
it's moving on the template

RT detection methods
Second method of
detection of PCR product
Detecting an increase in
fluorescence intensity
Utilize a fluorophore that
emits light only when
complexed with dsDNA
Increasing amount of
dsDNA product with each
round of amplification
gives a corresponding
increase in fluorescence
Fluorescence high
Fluorescence low
Fluorescence low
Fluorescence high
PCR Primers
Go to Genbank, look up sequence of virus or
organism of interest

PCR Primers
Info about publishers of sequence-when and who
Info about organism
Info about source of material


PCR Primers
Info about translation, capping, DNA sequence
PCR Primers
Identify gene sequence
in mRNA sequence



select primers to use for
RT and PCR

1 gtattaataa tgtcgacttc aggaactggt aagatgactc gcgcgcagcg tcgagctgcc
61 gctcgtagaa atcgtcggac cgctggggtc caaccagtaa ttgtcgaacc aatcgctgct
121 ggccaaggca aggccattaa agcgattgca ggatacagca tatcaaagtg ggaggcgtct
181 tcggacgcga ttacagcgaa agccaccaat gccatgagta tcactctgcc ccatgagctc
241 tcttctgaaa agaataagga gcttaaggtc ggcagagtgc tgctttggtt gggacttctt
301 cctagcgttg ctgggaggat taaggcttgt gttgctgaga aacaggcaca ggccgaggcc
361 gcttttcaag tagccttggc ggttgctgac tcctcgaaag aggtggtcgc ggccatgtat
421 acggacgcct ttcgaggggc gactctgggg gatttgctta atctccagat ttatctgtat
481 gcatctgaag cagtgcctgc taaggcggtc gttgtacatc tagaagttga gcacgtaagg
541 cctacgttcg atgacttctt caccccggtt tataggtagt gcccctgctc ggagagcccc
601 tgactgggtt aaagtcacag gccccttgtc tcaggtagag accctgtcca ggtaggacac
661 tttggctaag gttaaaagct tgttgaatca gtacaataac tgatagtcgt ggtttacacg
721 cagacctctt acaagagtgt ctaggtgcct ttgagagtta ctctttgctc tcttcggaag
781 aacccttagg ggttcgtgca tgggcttgca tagcaagtct tagaatgcgg gtaccgtaca
841 gtgttgaaaa acactgtaaa tctctaaaag agacca
1 gtattaataa tgtcgacttc aggaactggt aagatgactc gcgcgcagcg tcgagctgcc
61 gctcgtagaa atcgtcggac cgctggggtc caaccagtaa ttgtcgaacc aatcgctgct
121 ggccaaggca aggccattaa agcgattgca ggatacagca tatcaaagtg ggaggcgtct
181 tcggacgcga ttacagcgaa agccaccaat gccatgagta tcactctgcc ccatgagctc
241 tcttctgaaa agaataagga gcttaaggtc ggcagagtgc tgctttggtt gggacttctt
301 cctagcgttg ctgggaggat taaggcttgt gttgctgaga aacaggcaca ggccgaggcc
361 gcttttcaag tagccttggc ggttgctgac tcctcgaaag aggtggtcgc ggccatgtat
421 acggacgcct ttcgaggggc gactctgggg gatttgctta atctccagat ttatctgtat
481 gcatctgaag cagtgcctgc taaggcggtc gttgtacatc tagaagttga gcacgtaagg
541 cctacgttcg atgacttctt caccccggtt tataggtagt gcccctgctc ggagagcccc
601 tgactgggtt aaagtcacag gccccttgtc tcaggtagag accctgtcca ggtaggacac
661 tttggctaag gttaaaagct tgttgaatca gtacaataac tgatagtcgt ggtttacacg
721 cagacctctt acaagagtgt ctaggtgcct ttgagagtta ctctttgctc tcttcggaag
781 aacccttagg ggttcgtgca tgggcttgca tagcaagtct tagaatgcgg gtaccgtaca
841 gtgttgaaaa acactgtaaa tctctaaaag agacca

Primers
www.fisheroligo.com (operon)
scale price/base
50 nmole $0.50
200 nmole $1.00
25 base oligo =$12.50

www.idtdna.com
scale price/base
100 nmole $0.55
250 nmole $0.95
25 base oligo =$13.75

www.fisheroligos.com (sigma genosys)
scale price/base
50 nmole $0.75
200 nmole $1.30
25 base oligo =$18.75

Primers
Real-Time PCR Probes
Dual-labeled fluorogenic probes for
TaqMan real time quantitative PCR.
www.synthegen.com
60 nmol $500.00

Taqman probe:
High-quality primer of minimum cost
Economic PAGE/HPLC purification
www.genscript.com
50 nmol $120.00
Unknown ID
Barley came in with signs of viral infection

TEM analysis indicated a ~30 nm icosahedral particle in infected plant sap

Extracted virus from infected material
biochemical characterization

Extracted nucleic acid from virus preparation
reverse transcriptase to generate DNA copy of RNA
PCR sequence



Could have done RT PCR from nucleic acid extracted from plant via a RNA
extraction kit

Passage onto uninfected host

Unknown ID
genomic RNA extracted from pur. virus
CCMV
unknown (BMV)
SDS page of viral protein
CCMV
unknown (BMV)
Unknown ID
Dot blot of purified virus
a-CCMV
antibody
a-BMV
antibody
CCMV BMV CCMV BMV
RT-PCR with
BMV coat protein
specific primers
virus extracted RNA
BMV
CCMV
BMV
CCMV
size marker