![]() (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences: application to haemophilia A. (2000) The Nucleic Acid Protocol Handbook. ![]() (1985) Antenatal diagnosis and carrier detection of haemophilia A using factor VIII gene probe. (1985) Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene. (1995) Molecular genetics and counselling in haemophilia. (1993)Report of a joint WHO/WFH meeting on the control of haemophilia: carrier detection and prenatal diagnosis. P., Boulyjenkov, V., Briet, E., Chan, V., et al. TaqI is a restriction enzyme from the same type of organism that produces Taq DNA polymerase (Thermophilus aquaticus or Thermus aquaticus).White, G. However, there are exceptional temparatures: SmaI (25C),ApaI (30C), BclI (50C), BstEII (60C) and TaqI (65C). Most restriction enzymes are used at 37C. RE Digestion What does a restriction enzyme need in order to do its duty? - a double-stranded DNA sequence containing the recognition sequence.- suitable conditions for digestion. What type of ends produced by the REs? RE Specific Sequence (5’ 3’) Type of ends SalI HaeIII HhaI HpaI MboI NotI PstI XhoI XbaI SacI Tutorial Give the specific sequences recognized by the REs. They may have different sites of specific cleavage.Restriction enzymes that recognize the same sequence and are derived from different organisms.REs 4-base cutters 6-base cutters HpaII CCGG HindIII AAGCTT S S RsaI GTAC EcoRI GAATTC B S TaqI TCGA DraI TTTAAA S B AluI AGCT BamHI GGATCC B S HinfI GANTCěglI AGATCT S S DdeI CTNAGĜlaI ATCGAT S S B = blunt ends S = sticky ends (overhang) Sticky ends 5’G/CGC3’ 5’G CGC3’ CfoI 3’GCG/C5’ 3’CGC G5’ 5’GAT/ATC3’ 5’GAT ATC3’ Blunt ends EcoRV 3’CTA/TAG5’ 3’CTA TAG5’ Sticky ends 5’G/CGC3’ 5’G CGC3’ CfoI 3’GCG/C5’ 3’CGC G5’ 5’GAT/ATC3’ 5’GAT ATC3’ Blunt ends EcoRV 3’CTA/TAG5’ 3’CTA TAG5’ REs 4-base cutters 6-base cutters HpaII CCGG HindIII AAGCTT RsaI GTAC EcoRI GAATTC TaqI TCGA DraI TTTAAA AluI AGCT BamHI GGATCC HinfI GANTCěglI AGATCT DdeI CTNAGĜlaI ATCGAT Some REs produce blunt ends and others produce sticky ends. RFLP analysis has been widely used in genetic research, forensic investigations, and paternity testing. 4-base cutters 6-base cutters HpaII CCGG HindIII AAGCTT RsaI GTAC EcoRI GAATTC TaqI TCGA DraI TTTAAA AluI AGCT BamHI GGATCC HinfI GANTCěglI AGATCT DdeI CTNAGĜlaI ATCGAT Restriction Fragment Length Polymorphism (RFLP) analysis is a technique used to detect genetic variation by examining the lengths of DNA fragments produced by the digestion of DNA samples with specific restriction enzymes. over 3000 REs have been studied in detail, and more than 600 of these are available commercially.Two catalytic manganese ions (one from each monomer) are shown as magenta spheres and are adjacent to the cleaved sites in the DNA made by the enzyme (depicted as gaps in the DNA backbone). Structure of the homodimeric restriction enzyme EcoRI(cyan and green cartoon diagram) bound to double stranded DNA (brown tubes). BamHI (Bacillus amyloliquefaciens) HindIII (Haemophilus influenzae) TaqI (Thermus aquaticus).Iğirst identified (order identified in bacterium).Restriction enzymes are named based on bacteria in which they are isolated in the following manner:.Daniel Nathans and Hamilton Smith received Nobel Prize in Medicine (1978) for the discovery of restriction endonucleases, leading to the development of recombinant DNA technology.Then create a restriction enzyme that will cut any DNA at that sequence. DNA genome of pine tree restricted by EcoRI can generate 5 million different restricted fragments. Restriction Fragment Length Polymorphism.RE acts as molecular scissors to cut DNA molecules at specific sequence.RFLP was developed at the late 70’s due to the discovery of restriction enzymes (REs or called as restriction endonucleases) from bacteria.RFLP(Restriction Fragment Length Polymorphism)
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