BIO314H5 Lecture Notes - Lecture 7: Steric Effects, Restriction Digest, Wavelength
Document Summary
Using a single template strand which is sequenced with using normal ntps and also a mixture of ddntps which are similar in structure enough to the original bases to be incorporated into the growing dna chain. Many copies of different fragments lengths are generated in each reaction, terminated at the locations where the ddntps were inserted. Lots of fragments with different lengths: hence using electrophoresis to separate fragments based on size o. Dideoxy provide certain portion of dideoxy bases which cannot have other bases added to it: leads to termination here, have to get the termination of this dideoxy base exactly. Sanger sequencing original: four separate reactions to be done: then have to analyze every band to determine sequence, how to determine one bp difference in gel. Automatic sequencing : one reaction, get the proportions different, different label on each bases which incorporated in each fragment at the end, readout in a capillary.
