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MOLECULAR MAPPING

BY PCR-Sapate P D,

ABW/34/2011.

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INTRODUCTION

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POLYMARASE CHAIN

REACTION It is an in vitro amplification of template DNA using

a basic principle of “semi conservative replication of DNA” naturally found in living cell.

It is completely an automated reaction carried out a thermo cycler , utilizes following things-

• Template DNA

• Two oligonucleotide primers

• dNTP’s

• Heat stable DNA polymerase

It generates billion copies of DNA , present even as a single copy in the initial preparation, within few hours.

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PCR involves amplification of a specific gene

or DNA sequence within a very less duration.

Specificity is one of the important attribute

of PCR.

It generally involves following steps-Step Temperature(˚

C)

Duration Description

Denaturation 94 2 min Two strands of DNA

get separated

Primer

annealing

40-60 1 min Primer get joined to 3’

end of any strand

Extension 72 2 min New strand formation

at free 3’end of primer

by taq polymerase

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This is a single cycle for a few minutes.

In thermal cycler, this cycle is automatically

repeated for 30-40 times which requires few

hours to complete.

PCR has wide range of applications, one of is

‘creation of molecular(genome) maps’.

Previously biochemical & protein based

markers used but PCR makes revolutionary

change in this field.

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MOLECULAR MAPS

It may be defined as “a detailed structure &

functional organization of the complete genome of

an organism”.

The ultimate aim is to obtain the DNA sequences

of the complete genome , which provides most

detailed molecular description.

Initially , traits were observable with naked eyes

were used for mapping, then comes isozymes

markers. But due to there lack of availability in the

genome , molecular markers used to produce

genome maps.

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These were divided in following categories-

Genetic mapping:

It is an schematic representation of

various genetic markers in the specific orders

in which they are located in chromosome as

well as the relative distances between these

makers.

The distance is given in terms of

centiMorgan(cM) which are proportionate to

recombination frequencies.

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Physical mapping :

It uses molecular biology techniques to

examine DNA molecules directly in order to

construct maps showing the positions of

sequence features, including genes.

The distance shows actual number of base

pairs present within the two possible genes.

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Genetic map, serves to guide a scientist

toward a gene, just like an interstate map

guides a driver from city to city.

Physical maps mark an estimate of the true

distance, in measurements called base pairs,

therefore, allow a scientist to more easily

home in on the location of a gene.

Both genetic and physical maps provide the

likely order of items along a chromosome.

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Linkage analysis is the basis of

genetic mapping

Chromosomes are inherited as intact units, so it was reasoned that the alleles of some pairs of genes will be inherited together because they are on the same chromosome. This is the principle of ‘genetic linkage’.

Pairs of genes were either inherited independently, as expected for genes in different chromosomes, or, if they showed linkage, then it was only partial linkage: sometimes they were inherited together and sometimes they were not.

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HISTORY

1913- first linkage map was constructed in

drosophila melanogaster.

1985-PCR was developed by kary mullis.

1988- Olson et al suggested STS’s(sequence

tagged sites) for physical mapping of human

genome.

1989-kary mullis discovered thermo stable

polymerase (taq polymerase).

“Science” announced taq polymerase as

‘molecule of the year’.

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1993- Kleinhofs et al constructed first whole

genome linkage maps of barley based on

molecular markers (mainly RFLPs).

2006-A potato linkage map was generated

with over 10,000 AFLP loci by Van Os et al .

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WHY PCR USED IN

MAPPING?

Using gene as a marker is very useful but it has

limitation.

Gene occupy very small portion/space of

genome and are not evenly distributed in the

genome.

Also every gene not have allelic forms or can

not distinguishable easily.

Therefore the map based on gene is not

detailed.

So there comes need of other markers, which

is fulfilled by DNA markers(PCR based

markers).

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DIFFERENT PCR BASED

MARKERS USED FOR MAPPING

A molecular marker is polymorphic DNA sequence used for chromosome mapping.

A marker is said to be polymorphic if it has different forms in different individual of a species.

There were mainly 3 PCR based markers used to prepare genetic maps .

• RAPD

• AFLP

• STS

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RAPD

It is an Random Amplified Polymorphic DNA, which uses random/arbitrary oligonucleotide primers (pronounced as ‘rapids’).

The size of primer used ranges 2 – 10 bp which is not specific.

Genomic DNA has complementary sequences to RAPD primers to many location.

After PCR, in some strains which has complementary region detected in gel but those do not having region can not be detected.

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These bands constitute the ‘RAPD loci’.

RAPD marker is dominant marker i.e.

presence of band is dominant & absence of

band is recessive.

DNA bands of different sizes are assumed to

be amplified product of different RAPD loci in

the genome.

RAPD markers shows limited variation

between parents mainly in inbreeding species.

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AFLP

Amplified Fragment Length Polymorphism; a

molecular marker generated by a combination

of restriction digestion and PCR amplification.

The power of this procedure is that you can

generate a large number of map able loci with

a single amplification.

This will help you saturate a region of the

genome rather quickly.

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Thus marker allows the simultaneously

screening of a large number of anonymous

markers randomly distributed throughout the

genome.

The drawback is that procedure is a bit time

consuming.

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Sequence Tagged Sites

STS is simply a short DNA sequence generally

100 to 500 bp and occurs only once in the

chromosome or genome.

STS mapping is performed by multiple STS or

set of STS on broken/fragmented

chromosome/genome.

A collection of DNA fragments is made by

isolating a chromosome and then breaking it

into smaller pieces, so that in collection a single

point can be represented about five/six times.

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The mapping is performed by amplification of

STS unique sequence using PCR and looking

for the presence of two different STS on the

same fragment from the collection.

The frequency of having two STS on the same

fragment depends how close they are to each

other.

Closer the STS to each other higher the chance

to find them together on the more fragments ,Or

frequency at which breaks occur between two

markers.

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Any unique DNA sequence can be

used as an STS -

The STS can be any sequence which:

• Have known sequence

• Should have unique position in

chromosome/genome

The most common STS are:

• Expressed sequence tags (ESTs) (taken from

cDNA projects: limited to genes only)

• Simple sequence length polymorphism

(SSLPs) (mini and micro satellite )

• Random genomic sequences

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Comparison between markers

used in mapping

features RAPD AFLP STS

Efforts to generate

marker

Simple Simpler than

RFLP

Difficult

Reproducibility Moderate High high

Dominance Dominant Co dominant Co dominant

Information

generated

High Very high Very high

Time required for

detection

Short Longer than other short

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Importance of gene mapping

Gene map is the anatomy of genome. It is a prerequisite to understand functioning of genome.

Helps in analysis of the heterogeneity and segregation of genetic diseases.

Helps to develop methods for gene therapy.

Provides clinically useful information about linkage.

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REFRANCES

Elements of biotechnology by P K Gupta

Biotechnology by B D Singh

www.springer.com

www.ncbi.nlm.nih.gov (Bookshelf ID:

NBK21962)

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QUERY????? (IF ANY)

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