Biology 1002B Lecture Notes - Lecture 20: Sanger Sequencing, Pyrosequencing, Dideoxynucleotide
27 Jan 2020
School
Department
Course
Professor
Document Summary
Mental floss: pcr primer design if you wanted to pcr amplify only the coding region of this gene you would make the primer below: Outcomes: current trend in cost of dna sequencing. Decreasing fast: steps in typical dna sequencing methodologies. Get dna, purify it, get rid of proteins, rna. Break the dna up because cannot be sequenced from one end to another, has to be done in fragments. After sequencing it is assembled back: purification, fragmentation, amplification, sequencing fragments, assembly of fragment sequences, structural features of dideoxynucleotides. They do not have a 3"oh, instead have h on the 3" carbon so no further base can be added: basic mechanism of classical vs. automated sanger sequencing. Classical sanger sequencing involves 4 tubes, each tube has all four deoxynucleotides (datp, dttp, dgtp, dctp) and one of 4 dideoxynucleotides. Dna polymerase synthesizes the new strand until a dd nucleotide is incorporated which acts as a terminator.
