1. GENETIC MARKERS (RFLP, AFLP, RAPD, MICROSATELLITES, MINISATELLITES)
PRESENTED BY:
PARUL
M.SC. BIOCHEMISTRY
IIIrd SEMESTER

2. GENETIC MARKERS They are genes or DNA sequences with a
known location on a chromosome that can
be used to identify individuals or species.

3. Uses: They are used to identify different features in DNA sequence.
They can be used to differentiate between individuals in a population, or to classify individuals between different varieties or cultivars within a species.
The different features in the sequence can be used to identify if that particular region was inherited from the female or male parent.
It can be used to build up a more complete picture of each individuals genotype at each marker.
This also allows us to track the inheritance of different regions of the genome.
Some commonly used genetic markers are:
RFLP
SSR OR MICROSATELLITES
AFLP
RAPD
MINISATELLITES

4. Restriction fragment length polymorphisms (RFLPs)
RFLPs were the first type of DNA marker to be studied.
Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples with specific restriction endonucleases.
Restriction enzymes cut DNA molecules at specific recognition sequences.
This sequence specificity means that treatment of a DNA molecule with a restriction enzyme should always produce the same set of fragments. But when there is a polymorphic restriction site present in different allele of chromosome then DNA fragments of different length are produced.

5. WORK FLOW: Large scale genomic DNA isolation
Digest with selected restriction enzyme
Gel electrophoresis
SOUTHERN BLOT- transfer fragments to nitrocellulose membrane filter
Hybridisation using radio labelled probe
Detect probe signal

6. RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)
RAPD markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.
It do not require any specific knowledge of DNA sequence of target organism.
The primers will or will not amplify a segment of DNA depending on positions that are complementary to the primers’ sequence.
Therefore, if a mutation has occurred in template DNA at site that was previously complementary to primer, a PCR product will not be produced. This results in different pattern of amplified DNA segments on gel.

7. FIG 2. RAPD ANALYSIS
FIG 1. WORK FLOW OF RAPD

8. Amplified fragment length polymorphism (AFLP)
These are the differences in restriction fragment lengths caused by SNPs (single nucleotide polymorphism) or INDELs (insertion or deletions) that create or abolish restriction endonucleases recognition sites.
The AFLP technique is based on selective PCR amplification of restriction fragments from total digest of genomic DNA. It is a hybrid of RAPD and RFLP techniques.
Typically, two different restriction enzymes are used to cut the genomic DNA to produce large number of fragments.
Restricted fragments are ligated with adapters (25-30 bp long). Primers are used to amplify the restricted fragments. These primers are complementary to adapters.
The primers used vary at 3’-end such that they will amplify only a subset of restricted DNA fragments. Later, polymorphic or monomorphic bands are analysed as in RAPD.

9. WORK FLOW:
Digestion of total cellular DNA with one or more restriction enzymes.
ligation of restriction half-site specific adaptors to all restriction fragments.
Pre-amplification with two primers that have corresponding adaptor and restriction site specific sequences.
Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site more specific sequences.
Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

10. MICROSATELLITE DNA
Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences.
They are also called SSR, i.e., short sequence repeats bp tandemely repeats are called microsatellites.
They tend to occur in non-coding regions of the DNA (this should be fairly obvious for long dinucleotide repeats).
Although a few human genetic disorders are caused by (trinucleotide) microsatellite regions in coding regions.
On each side of the repeat unit are flanking regions that consist of "unordered" DNA.
The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction).
The repeats vary in different samples but flanking regions where PCR primers bind are constant.

11. Advantages of microsatellites as genetic markers:
Locus-specific (in contrast to multi-locus markers such as minisatellites or RAPDs) 
Co-dominant (heterozygotes can be distinguished from homozygotes, in contrast to RAPDs and AFLPs) 
PCR-based (means we need only tiny amounts of tissue; works on highly degraded or "ancient" DNA) 
Highly polymorphic ("hypervariable") -- provides considerable pattern 
Individual alleles at a locus differ in number of tandem repeats of unit sequence owing to gain or loss of one or more repeats and they can be differentiated by electrophoresis according to their size.

12. MINISATELLITE DNA Unit - 14-500 bp (average about 20).
Repeat - Generally times ( bp long).
Location - Generally euchromatic.
Examples - DNA fingerprints. Tandemly repeated but often in dispersed clusters. Also called VNTR’s (variable number tandem repeats).
Minisatellites demonstrate intraspecies polymorphism and this been used for DNA fingerprinting of organisms
When cloned probes containing a minisatellite sequence are annealed with DNA blots containing restriction endonuclease digests of DNA, multiple bands hybridize. The pattern of bands varies from one individual of a species to another but is the same when DNA from several tissues of a single individual is examined.

13. RFLP RAPD AFLP Microsatellite and Minisatellite Pros:
High reproducibility
Show co-dominant alleles
Reliable marker in breeding analysis
Cons:
Require large quantity of DNA
Expensive process
Time consuming
Labor intensive
Pros:
Quick and easy
Low quantity of DNA required
Inexpensive
Do not require any specific knowledge of target
Cons:
Low reproducibility
Highly sensitive and complicated procedure
PCR conditions influence outcome
Mismatches between primer and template may result in complete absence of PCR product
AFLP
Microsatellite and Minisatellite
Pros:
High genomic abundance
Considerable reproducibility
Higher resolution and sensitivity
Cons:
Need for purified DNA
Dominant markers
Abundance of data
Pros:
Low quantity of template DNA required
High genomic abundance
Co-dominant marker
High reproducibility
No high quality DNA is required
Cons:
High development cost
Errors in genotyping scoring
Difficulty in interpretation
PCR error

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