-RFLP, -RAPD -AFLP,

Genetic Marker

1. Classical

markers 2. DNA/Molecular markers

-Morphological, --cytological and biochemical markers

-RFLP, -RAPD -AFLP, -SSRs, -SNP

Genetic marker

The genetic marker is a gene or DNA sequence with a known location on chromosome controlling a particular gene or trait.

 Genetic markers are closely related with the target gene and they act as sign or flags.

 Genetic markers are broadly grouped into two categories:

2. DNA marker OR Molecular markers

molecular marker is present, since the marker indicates the presence of

the desired characteristic.

Individuals within a population of a sexually reproducing species will have some degree of heritable genomic variation caused by mutations, insertion/deletions (INDELS), inversions, duplications, and translocations. Such variation can be detected and screened using molecular, or genetic, markers. By definition, molecular markers are genetic loci that can be easily tracked and quantified in a population and may be associated with a particular gene or trait of interest.

Properties of molecular markers

An ideal DNA makers should however poses the following properties.

(i) Highly polymorphic

(ii) Co dominant - different form of marker should be detected in a diploid organism to allow discrimination of homozygote and heterozygote.

(ii) Frequent occurrence in genome

(iii) Selective neutral behaviour (the DNA sequences of any organism are neutral to environmental conditions or management practices). Therefore, selection based on molecular marker will be highly efficient and would be independent of environment

(iv) Easy access (availability)

(v) Easy and fast assay

Types of DNA Markers

1. Non-PCR based genetic markers (Restriction fragment length polymorphism)RFLP

 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 in question with specific restriction endonucleases.

 Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.

 An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis

 How It Works

electrophoresis and blotting. Blotting patterns are unique to each organism and characterize the specific genotypes.

Steps in RFLP

1. Isolation of sufficient amount of DNA from samples

2. Fragmentation of the DNA samples with specific restriction endonucleases into short sequence

3. Separation of the resulted fragments with different lengths by agarose gel electrophoresis.

4. Transfer of the gel profile into a membrane by Southern blotting

2. Randomly amplified polymorphic DNA (RAPD)

 RAPD is a useful molecular marker in molecular biology. It is a quick and easy technique.

 RAPD can be defined as a method which results in polymorphic DNA sequences as a result of random amplification of multiple locations of the target DNA template.

 RAPD uses short oligonucleotide primers with arbitrary sequences for the PCR amplification. Randomly amplified polymorphic DNA, or RAPD, marker analysis utilizes short PCR primers consisting of random sequences usually in the size range of 8 to 15 nucleotides in length.

 RAPD MARKERS ARE NOT LOCUS SPECIFIC Primers are artificially synthesized without the prior sequence knowledge. Hence, it is considered as an easy and useful technique.

 Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies)

The following major steps are involved in RAPD.

1. Extraction of target DNA

2. Amplification of the multiple locations of the target DNA using randomly chosen primers

3. Gel electrophoresis of the amplified PCR products

4. Staining with ethidium bromide and identification of the polymorphism

What is the difference between RAPD and RFLP?

RAPD is a molecular marker based on random

primers and PCR. RFLP is a molecular marker based on the production of different length restriction fragments.

Required Sample Small DNA samples are enough for the RAPD

analysis. A large amount of extracted DNA sample is required for RFLP analysis.

Time

RAPD is a quick process. RFLP is a time-consuming process. Primer Use

Random primers are used and same primers can be

used for different species. Species-specific probes are used in RFLP for hybridization.

Reliability Reliability of the technique is less compared to

RFLP.

RFLP is a reliable technique.

Blotting

RAPD involves southern blotting. Southern blotting is a one step of RFLP. Detection of Allelic Variation

Allelic variations cannot be detected by RAPD. Allelic variations can be detected by RFLP. Need for Sequence Knowledge

RAPD does not require prior sequence knowledge. Prior sequence knowledge is required for probe designing. PCR

PCR is involved with RAPD PCR is not involved with RFLP. Reproducibility

RAPD has a low reproducibility RFLP is has a high reproducibility compared to RAPD.

Suggested Reading

https://slideplayer.com/slide/5137/