this ppt will help you to study PCR based markers used

1. 1
9/30/2014

2. MOLECULAR MAPPING
BY PCR
-Sapate P D,
ABW/34/2011.
2
9/30/2014

3. INTRODUCTION
3
9/30/2014

4. 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.
4
9/30/2014

5.  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
5
9/30/2014

6.  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.
6
9/30/2014

7. 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.
7
9/30/2014

8.  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.
8
9/30/2014

9.  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.
9
9/30/2014

10.  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.
10
9/30/2014

11. 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.
11
9/30/2014

12. 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’.
12
9/30/2014

13.  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 .
13
9/30/2014

14. 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).
14
9/30/2014

15. 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
15
9/30/2014

16. 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.
16
9/30/2014

17.  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.
17
9/30/2014

18. 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.
18
9/30/2014

19.  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.
19
9/30/2014

20. 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.
20
9/30/2014

21.  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.
21
9/30/2014

22. 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
22
9/30/2014

23. 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
23
9/30/2014

24. 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.
24
9/30/2014

25. REFRANCES
 Elements of biotechnology by P K Gupta
 Biotechnology by B D Singh
 www.springer.com
 www.ncbi.nlm.nih.gov (Bookshelf ID:
NBK21962)
25
9/30/2014

26. QUERY????? (IF ANY)
9/30/2014
26

27. 27
9/30/2014