2. Molecular markers:
The variant DNA fragment that
gives some information about genes of
interest is called molecular
marker/genetic marker.
3. Types of molecular marker:
1.PCR based marker.
RAPD.
2.Hybridization based markers.
RFLP.
AFLP.
SNP.
4. Restriction fragment length polymorphism:
The variation in the restriction DNA
fragment lengths between individuals of a
species is called restriction fragment length
polymorphism. This is a best laboratory
technique to analyze and compare DNAs of two
or more individuals of a species. It is
extensively used in genome analysis.
5. RFLP method involves the following steps:
Sample collection.
Isolation DNA.
Restriction digestion.
Electrophoresis.
Blotting of DNA.
Making genomic DNA probes.
Nucleic acid hybridization.
Autoradipgraphy.
6. sampe collection:
Tissues or cells of individuals are collected to extract their
DNA. The samples are collected separately.
Isolation of DNA:
The DNA was isolated from the tissues or cells.
restricton digestion:
Genomic DNA of each sample is cut with a restriction
enzymes separately to generate variable lengths of DNA
fragments.restricion enzymes such as EcoRI .Hind III, PstI are of
much use for RFLP. This restriction digest is divided into two halves
one half is used for DNA DETECTION and other is used for PROBE
MAKING.
7. Electrophoresis:
The digested genomic DNA of all the samples are loaded
into separate wells in agarose or polyacrylamide gel and are
subjected to electrophoresis. The DNA fragments get separated
according to their size .use of molecular weight markers in one lane
is likely to know the molecular weight of separated DNA fragments.
Blotting of DNA :
As in southern blotting the DNA is transferred to a
nitrocellulose filter by using a blotting setup. The nitrocellulose filter
is dried in between filter discs before performing hybridization.
8. Making genomic DNA probes:
One half genomic DNA digest from each sample is
electrophoresed to separate the DNA fragments. Fragments of 0.5-
2.0 kb are extracted from the gel and cloned in Puc21 vector to
construct rDNAs.
These rDNA are amplified by introducing them into bacterial
host and the amplified rDNAare isolated from the bacteria . the
target DNA fragments are excised from the rDNA by using the
same restriction enzyme that was Used for cutting the DNA. They
are purified by electrophoresis.
9. These DNA fragments are radiolabelled with p35 isotope
incorporated nucleotide by nick translation or random primer
extension or end labeling. As a result, genomic probes are
formed
Nucleic acid hybridization:
DNA blotted nitrocellulose membrane is kept immersed
in a hybridization solution containing genomic DNA probes to
bring out hybridization. After hybridization unbound probes are
washed out of the membrane.
10. Autoradiography:
Images of radioactive probes are captured on an X-ray
film using autoradiography. The autoradiogram shows DNA
bands in distinct lanes each of which is characteristics of an
individual. Variation in the lengths of DNA fragments between
individuals as shown by the lanes in the autoradiogram is called
RFLP.
13. Random amplified polymorphic DNA :
The RAPD is a PCR based method to detect
variations between individuals of a species by
selective amplification of some polymorphic
sequences in their genome. this method was
developed by J.G.K.WILLIAMS in 1991. only
least number of DNA fragments are considered for
RAPD analysis. RAPD are of much use to
construct genetic maps.
14. Steps involved in RAPD analysis:
1.Sample cells or tissues are collected from individuals to be
distinguished from one another.
2.Genomic DNA of each and every sample is isolated by using a
standard procedure.
3.each genomic DNA is separately treated with Taq polymerase, a
primer ,Datp,dttp,dctp,dgtp.the primer is 9-10 bp
4. all these reaction mixture are subjected to repeated cycles.
15. denaturation,primer annealing, polymerization upto 35-40 cycles.
Denaturation: temperature is maintained at 940 c for 1 mins
Annealing:360c for 2 mins
Polymerization: 720c for 1.5 mins
Repeated cycle of denaturation, annealing, polymerization leads to
amplification of polymorphic sequences in the genome.
5.As a result of PCR amplification each reaction tube contains RAPD
fragments. Ethidium bromide is added to it.
6.The RAPDs thus obtained from the samples are separately loaded
into wells of an AGE or PAGE and molecular weight markers are
added on the well.
16. 7.The loaded DNAs are electrophoresed to separate the DNA
fragments based on their size.
8.The electrophoresed gel is examined under a UV light illuminator to
detect light bands and photographed.
If a DNA band of molecular weight 1Kb distinguish an
individual from others it is considered as RAPD marker of that
individual. An individual may differ from others in one or more
RAPD markers.
17.
18. Advantages:
There is no need for species specific probes in RAPD analysis.
RAPD is quick method.
RAPD analysis can be performed in crude DNA samples also.
RAPD requires only small amount of DNA samples.
It does not require radioactive probes and hybridization.
