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Day 10 Evening Lecture Notes

Steve Williams, Smith College

June 15, 2004

Given 600-800 bp sequences from a single gel, how do we perform additional reassembly? We need appropriate primers to generate many overlapping sequences.

Obtaining sequencing primers

STRs are very hard to sequence as primers will find multiple binding sites. Centromeres, telomeres and LINE1 all have this problem. Two LINE1 repeats are typically about 90% the same. Should this expensive sequencing even be performed?

cDNA library sequences are often put into databases after 1 pass of sequencing and are often unreliable. For new sequences GenBank will require sequencing of both strands.

Primers shouldn't be designed to hybridize to the very end of an unknown sequence because the data quality there is always very poor.

Most sequence nowadays are run with thermal cycling, bases analogues and denaturing gels, all the tricks at once. Some companies now will even prepare a sequence from a colony on a gel without any further preparation by the researcher. Such facilities usually ask to see a quantitation gel photo first. A general rule in molecular biology is to perform lots of controls and checks all the time as doing so saves effort in the long run.

Discussion with Barton Slatko about the Sargasso Sea experiment

How did Venter's group obtain a species count from such a mishmash of DNA fragments from so many species? By analyzing ribosomal DNA sequences that are highly conserved over evolution but slightly different from species to species. Primer design and reassembly are therefore relatively straightforward for these species. From the ribosomal sequences some further walking backward and forward on the genomes could be done, but sequencing the whole genomes of the organisms was not the point.

Molecular biological ecosystem surveys are becoming more common. Some companies like Diversa specialize in looking for new restriction enzymes in the field. People are trampling all over Yellowstone and the Antarctic looking for new extremophiles. A colleague of Bart's sequenced his own septic system and found two new restriction enzymes.

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