PCR with Sequence Specific Primers

PCR with Sequence Specific Primers
  • An approach opposite to the PCR with arbitrary primers is to design primers to target specific genome. These primers are based on sequence specific knowledge of the genome and bind to a particular complementary sequence because of their long length and specificity.
  • Sequence Tagged Sites (STS)
  • Sequence tagged sites are primers that are based on some degree of sequence knowledge of genome under study. Sequence tagged site primers target and amplify a specific region of the genome. A sequence tagged site is general term given to a marker defined by its primer sequences amplified from the arbitrary primers.
  • Example of STSs are
  • Sequence Tagged Microsatellites (STMs)
  • Sequence Characterized Amplified Region (SCAR)
  • Cleaved Amplified Polymorphic Sequence (CAPS)
  • Only a limited quantity of DNA is required for this process.
  • highly polymorphic in nature
  • Good analytical resolution and high reproducibility because of the longer primer sequences.
  • Codominant in nature that is useful in distinguishing between homozygotes from heterozygotes.
  • Evenly distributed throughout the genome.
  • One obvious drawback of SSR marker is the development of polymorphic SSR marker because time, cost and skill is required for construction and screening of library and then sequencing of the hybridized clone to determine the flanking region of the repeats for SSR primer designing.
Sequence Characterized Amplified Regions (S CAR)
  • A technique in which RAPD marker termini are sequenced and longer primers are designed (15-30bp long) for specific amplification of a particular locus.
  • PCR products of SCAR produce a clear banding pattern then RAPDs.
  • Usually dominant but can be converted into codominant markers that can be used for genetic mapping.
  • There is no need to design and screen a library to pick up a sequence for primer synthesis, but sequencing efforts are required to find out the end sequences of RAPD products to synthesize specific primers for amplification.
  • So skill, efforts and expenses are required to design specific primers for each locus.
Cleaved Amplified Polymorphic Sequence (CAPS)
  • If specific primers designed for STMS, EST and STMS do not show polymorphism, than the amplified products are digested with a particular restriction enzyme and scored for polymorphism which is termed as Cleaved Amplified Polymorphic Sequences.
  • Codominant in ‘nature’ so the information can be effectively used for genetic mapping.
  • CAPSs are reproducible because of specific and long primer sequences.
  • To design a primer some genomic sequence specific knowledge is required.
  • Efforts and expenses are required to design specific primers for each locus.

Last modified: Monday, 2 April 2012, 11:26 PM