Primers and Probes

A brief introduction of Primer and Probes

1. Primer and Probes
Sunil Bhandari
BalkumariCollege
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2. Primer
•Aprimerisashortnucleicacidsequencethatprovidesastarting
pointforDNAsynthesis.Inlivingorganisms,primersareshort
strandsofRNA.Aprimermustbesynthesizedbyanenzyme
calledprimase,whichisatypeofRNApolymerase,before
DNAreplicationcanoccur.
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3. In sense of artificial purpose (Laboratory use)
What is Primer?
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4. Types of Primer..
A.Forward Primer: It attaches to the start codon of the
template DNA (the anti-sense strand)
B.Reverse primer: It attaches to the stop codon of the
complementary strand of DNA (the sense strand). The 5' ends
of both primers bind to the 3' end of each DNA strand
(Sense strand)
(Anti-Sense strand)©[email protected]

5. Role of Primer…….(initiate the Polymerization reaction)
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12. Primer design: Manual
•Take 18-24 sequences
•For forward primer, take sequence as it is and for reverse
primer, reverse complement the sequence
http://www.bioinformatics.org/sms2/rev_comp.html
•Include start codon (ATG) (in case of forward Primer and
Remove stop codon (TGA, TAA, TAG)in case of reverse
Primer
•Tm temperature of two primers should not vary more
than 5.
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16. Molecular Probes
•DNAorRNAprobesarestretchesofsingle-strandedDNA
orRNAusedtodetectthepresenceofcomplementary
nucleicacidsequences(targetsequences)by
hybridization.
•Probesareusuallylabelled,forexamplewith
radioisotopes,epitopes,biotinorfluorophorestoenable
theirdetection.
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17. Role of Probe……
•Mostly,Probesarelabelledwithradioisotopesoranyotherfluorescencedyes.
•Whenmixedwithtarget(template)DNA/RNA,probesdetectsthecomplementary
sitesintemplateandbindstoit.
•Thisprobe-templatebondedstateiscalledhybridizedconditionandprocessiscalled
hybridization.
•WhenProbebindtothetarget,labelledtheradio/fluorescentdyesemitsthesignal
whichcanbedetectedanditensurethehybridizationordetectionoftarget
part/sitesintemplateDNA/RNA.
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18. Probes vs Primer
•Probeisusedtodetectthe
presenceofaspecificDNA
fragmentinthemixture
through the
hybridizationwithadouble-
strandedDNA.
•Primer isused in
theinitiationofthe
polymerasechainreaction
byhybridizationwithsingle-
strandedDNA.
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