pcr & primer desging

8/7/2019 pcr & primer desging

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Dr. C.V.S.Siva Prasad BITS, Pilani,India

In situ Hybridization

In situ Hybridization

In general, nucleic acid hybridization isbased on the principle that

complementary sequences (DNA orRNA) will base pair with each otherto form a stable molecule

This principle is used in variety ofbiotechnology protocols includingsequencing, PCR, and Southernblotting


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Southern

Blotting

Southern

BlottingDNA is cut with restriction nucleases

and separated by gel electrophoresis.

DNA is denatured within the gel andtransferred (blotted) to nitrocelluloseor nylon membranes.

Labeled DNA probes are allowed to

hybridize to the blot.Labeled bands are visualized by

autoradiography.


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Polymerase Chain Reaction Amplify a single piece of DNA

Makes a large number of copies

Target specific primers allow specific

sequences to be amplified


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DNA Polymerase


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PCR

The Polymerase ChainReaction


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Molecular Biology ofPCR

The fundamental Molecular Biology of PCR is not

well understood.

We know what happens in a descriptive sense, but

not the physical chemistry/thermodynamics The rules for choosing PCR primers are a rough

combination of educated guesses and old fashioned

trial-and-error.

None of the published formulas for calculating

annealing temperatures has been proven to give

better than a rough estimate


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The PCR Process

In a nutshell, PCR works like this:

DNA and two primers are combined in a salt solution with

dNTPs and a heat stable DNA polymerase enzyme

The primers match some sequence in the target DNA

The solution is rapidly heated to DNA denaturing

temperatures (~95C) and cooled to a temperature where the

polymerase can function

Each thermal cycle generates copies of the sequence

between the primers, so the total number of fragments

amplifies in an exponential fashion: 2, 4, 8,16, 32, 64, etc.


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Primer Design Rules

primers should be at least 15 base pairs long

have at least 50% G/C content

anneal at a temperature in the range of 50-65degrees C

Usually higher annealing temperatures (Tm)

are better(i.e. more specific for your desired target)

forward and reverse primer should anneal at

approximately the same temperature


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GCGPRIME

The GCG program PRIME is a good tool for the designof primers for PCR and sequencing

For PCR primer pair selection, you can choose a targetrange of the template sequence to be amplified

In selecting appropriate primers, PRIME

allows youto specify a variety of constraints on the primer andamplified product sequences. upper and lower limits for primer and product melting temperatures

primer and product GC contents.

a range of acceptable primer sizes a range of acceptable product sizes.

required bases at the 3' end of the primer (3' clamp)

maximum difference in melting temperatures between a pair of PCRprimers


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Other Features ofPRIME

PRIME uses a simulated annealing test tocheck individual primers for self-

complementarity and to check the two

primers in a PCR primer pair forcomplementarity to each other.

Using this same annealing test, PRIME

optionally can screen against non-specificprimer binding on the template sequence and

on any repeated sequences you specify.


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Primer Design on the Web

There are a bunch of good PCR primer designprograms on the web:

Primer 3 at the MIT Whitehead Institute

http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

Cassandra at the Univ. of Southern Californiahttp://www-hto.usc.edu/software/procrustes/cassandra/cass_frm.html

GeneFisher by Folker Meyer & Chris Schleiermacherat Bielefeld University, Germany

http://bibiserv.TechFak.Uni-Bielefeld.DE/genefisher/

Xprimer at the Virtual Genome Center, Univ. MinnesotaMedical School

http://alces.med.umn.edu/rawprimer.html


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PCR for Medical Diagnostics


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PCR for Genetic Analysis

Ethical IssuesA number of newly discovered genes or

mutations of genes are correlated with

diseases such as cancer.PCR combined with DNA sequencing can beused to identify individuals with thesegenes.

Should this analysis be performed for adisease without a cure?Who should have access to this information?


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Diagnosis of Sickle-Cell

Anemia (by PCR)


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PCR for Cloning


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Dr. C.V.S.Siva Prasad BITS, Pilani,

India


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India


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India


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India

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