Genomic sequencing strategies

– 2 major strategies:

1. prepare genomic library (~15-fold coverage of genome)

(e.g. 100-300 kb inserts in BACs)

– less rearrangements

– map to find order, overlaps

– restriction maps, chromosome walking techniques

– map clones with minimal overlaps

– fewest clones needed

– e.g. sequence-tagged connectors (STC)

– sequence ends of all BACs

– use unique sequence from 1 clone

– find overlapping clones

– restriction map

– find clones with minimal overlaps

– or sequence-tagged sites (STS)

– unique regions amplified by PCR

– find clones with fewest of same STS

– minimal overlap

– completely sequence chosen clones

– shotgun clone fragments of BAC into M13 vectors

–>5-fold duplication of sequence (~3,000 clones)

– sequence individual clones

– assemble map from sequence overlap

– assemble each sequence into larger map by overlap

Advantage: minimizes duplication of sequence efforts

Disadvantage: mapping clones takes lots of time

2. newer strategy

– random shotgun clone entire genome into M13 vectors

– ~60 million clones for human genome

– assemble into overlapping sequence by computer analysis

Advantage: generates large amounts of sequence fast

Disadvantage: fragmentary sequence

– massive computer job to assemble

– but simplified by identifying known markers in sequence

– e.g. genes, BACs, STS

– allow sequences to be mapped to known sites

shotgun cloning (for either strategy above)

– generate library of ~0.8-1.5 kb M13 vector clones

–with >5-fold duplication of desired sequence

– randomly distributed through sequence

1. ligate ends of large DNA fragments (~100 kb)

– stops ends from being underrepresented in library

2. generate random fragments

Methods:

– shearing – break DNA up physically

– using sonication (high speed vibrations)

– or nebulization

(force DNA solution through high pressure siphon)

– droplets contain sheared DNA

Advantages: totally random fragments

– independent of sequence

Disadvantages – DNA ends frayed & uneven

– repair with T4 DNA polymerase & blunt end clone

– DNase I cleavage – endonuclease

– cleaves (fairly) randomly in DNA sequences

– partial restriction endonuclease cleavage – e.g. CviJI

– cleaves at PyGCPy and PuGCPu – blunt ends

– sites ~32 bp apart (on average)

– blunt ends for cloning

3. clone in M13 vectors – clones in both directions

4. sequence and align on computer to find overlaps