Hybridization based markers

Hybridization based markers
 
  • Hybridization based marker technology use cDNA, cloned DNA elements or synthetic oilgonucleotides as probes, which are labeled with radioisotopes or with conjugated enzymes that catalyze a coloured reaction to hybridize DNA.
  • The DNA, either cleaved with restriction enzyme or amplified by PCR, are separated by gel electrophoresis and transferred to a solid support matrix by Southern hybridization and hybridized mainly with labeled probe of known origin and sequence.
  • RFLP is representative of this type of technology. Restriction polymorphism occurs when mutation remove an existing restriction site or create a new restriction site. These alterations are detected by using a labeled probe

Restriction Fragment Length Polymorphism (RFLP)
  • RFLP was the first technique which enables the detection of polymorphism at the DNA sequence level. In this method, DNA is digested with restriction enzymes that cut the DNA at specific sequences, electrophoreed, blotted on a membrane and probed with a labeled oligonucleotide.
  • The DNA sequence variation detected by this method was termed restriction fragment length polymorphism.
Advantages
  • RFLP are co dominant markers, enabling heterozygote to be distinguished from homozygote.
  • The method is simple and no sequence specific information is required.
  • They are reliable markers in linkage analysis and can be easily determined if linked
  • Trait is present in a homozygous or heterozygous state in individual, an information highly desirable for recessive traits.
  • This technique has been successfully employed for tagging gene of economic value.
Limitations
  • Utility of RFLP has been hampered due to the large amount of DNA required for
  • restriction digestion and southern blotting.
  • The requirement of radioactive isotope makes the analysis relatively expensive and
  • hazardous.

Last modified: Monday, 2 April 2012, 11:17 PM