测序及建库专题.ppt

Sequencing Two ways for sequencing: 1. DNA molecules (radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. (Maxam and Gilbert chemical method, not widely used) 2. Chain-termination method (Sanger’s method, widely used) Chain-termination method ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end Shotgun sequencing of a bacterial genome The shotgun strategy permits a partial assembly of large genome sequence Assembly Step 1: form contigs (A single contig is about 50,000 to 200,000 bp. ) Sophisticated computer programs have been developed that assemble the short sequences from random shotgun DNAs into larger contiguous sequences called contigs. Assembly Step 2: The paired-end strategy permits the assembly of larger scaffolds (1-2 Mb) Fig 20-17. Contigs are linked by sequencing the ends of large DNA fragments (plasmid library containing larger DNA fragments). Genome-wide analysis The purpose of this analysis is to predict the protein coding genes (蛋白质编码基因) and other functional sequences (其他功能序列) in the genome. cDNA library generation, sequencing and application: The mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the cDNA library Sequence the cDNAs using shotgun method to generate EST (expressed sequence tag) database. These ESTs are aligned onto genomic scaffolds to help us identify genes and to assemble larger scaffolds. DNA library (DNA 文库) Genomic DNA library construction cDNA (complementary DNA) library construction Clone CDNA into a plasmid Treatment of cDNA with S1 nuclease (to remove possible 5 cap mRNA fragment remaining in cDNA duplex Convert potential ragged ends to blunt by treatment with Pol I (will fill in 5 overhangs and chew back 3 overhangs) Methylate cDNA at potential internal Eco RI sites by treatment with Eco RI methylase (plus S-adenosyl methioni