A linear map of different chromosomes of a species depicting the location of various molecular markers is known as molecular map.
Uses of Molecular Markers:
Varietal and hybrid identification 2. Identification of MS line of sunflower red leaf petiole (anthocyanin) pigment. 3. Identification of TGMS line in rice.
PTGMS lines with receptive purple leaf colours. 4. Identification of cultivars resistant to pests and diseases. Trichomeless trait in Cumbu – smut resistant Cotton-replant colour, hairiness of leaf glanded cotton offer resistance to various pests and disease. 5. Quality: Flax – varigated seed colour mutant used to identify fatty acid profile.
Low linoleic acid varieties can be developed through this.
Groups of Molecular Markers:
1. DNA hybridization based assays. i. Restriction Fragment length Polymorphism (RFLP) ii. Fluorescent In situ hybridization (FISH) 2. PCR based techniques using arbitrary primers i.
Random Amplified polymer DNA (RAPD) ii. DNA Amplification Fingerprinting (DAF) iii. Amplified Fragment Length Polymorphism (AFLP)
Steps in RAPD:
1. Isolation of DNA: i.
Powder the leaf in Liquid Nitrogen. ii. Keep it in extraction buffer at 65°C for 1 hour. iii.
Add equal volume of chloroform: isoamyl-alcohol (24:1). iv. Take aqueous phase and precipitate with isopropanol. v. Wash with 70% alcohol.
vi. Dry it and dissolve in ÒÅ buffer. 2. Purification of DNA: i. RNase treatment. ii.
Phenol: chloroform: Isoamylalcohol (25:24:1) treatment iii. Chloroform: Isoamylalcohol (24:1) treatment. iv.
Precipitate with isopropanol. v. Dry and dissolve in ÒÅ buffer. 3. PCR Amplification: i. Ten random primers will be used.
ii. PCR product will be resolved on 0.8% agarose gol. 4. Polymerase Chain Reaction (PCR): American biochemist Mullis, K.invented PCR and won Nobel Prize for same. Principle: Selective amplification of required region of DNA molecule.
Steps: 1. Denaturation of template DNA duplex by heating at 94°C. 2. Annealing of oligonucleotide primers to the target sequences of separated DNA strands at 35-65°C. 3. DNA Synthesis from the 3’-OH end of each primer by DNA polymerase.
Merits of RAPD:
i. High degree of polymorphism. ii. Minimum quantity of DNA is required. iii. Rapid analysis iv. Inexpensive compared to other methods.
Applications of PCR:
PCR can make use of very small amount of DNA. Even DNA in nanogram quantity can be easily handled. In view of this, PCR has many applications. Some of them are: 1.
Remnants of extinct organisms is sufficient to recover their DNA and amplify specific segments using PCR. Using this technique, from the remnants of woolly mammoth which lived about 40,000 years ago specific DNA segments were recovered and amplified. Now it is proved that this animal is related to present day elephant. 2.
PCR has revolutionised pre-natal detection of several genetic diseases. Only a very small sample of the foetus is sufficient to perform this work. Some of the diseases detected are: Phenylketonuria, cystic fibrosis, haemophillia A, beta thalassemia, sickle – cell anemia.
3. The sex of the invitro fertilised egg can be determined using PCR. The presence of the Y-chromosome can be detected at the 6-10 cell cleavage embryo without affecting the life of the embryo. 4. The PCR has wide and significant use in forensic science also. Small pieces of tissue, even a single hair left at the scene of the crime or one sperm lingering after a rape is enough to fix the identity of the criminal. 5. One can pick out a single antibody producing cell, amplify the genes encoding the antibody using PCR and clone them in bacteria to produce an anti-body producing bacteria.
Thereafter, any amount of antibody can be made by growing such bacterial cells in a fermenter.