BSC 315 Lecture Notes - Lecture 25: Reading Frame, Nucleic Acid Hybridization, The Terminal
14 Apr 2017
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In vivo dna synthesis: single stranded template, rna primer, dntps, dna polymerase. Dna synthesis in vitro proceeds 5" to 3". Sanger sequencing employs the biochemistry of dna synthesis. A sequencing reaction (in vitro) contains: dna polymerase, ssdna template, dna primer complementary to a short sequence in template, normal 2"-deoxynucleotides, also, some 2",3"-dideoxynucleotides. Normal dna nucleotides: 2"-deoxy- atp, ctp, gtp and ttp. 2" and 3" carbon (have h instead of oh). Absence of the 3" oh terminates dna synthesis because next nucleotide cannot be added (dna poly requires a 3" oh to add next base to; cannot join to 3" h. In sanger sequencing, every newly-made strand ends with a ddntp. The terminal base can be identified because each ddntp is tagged with a different fluorescent dye. A dna sequencing reaction: single-stranded template, dna primer. Primer is synthesized by methods of organic chemistry.
