Genomic libraries

2) Fragmentation of DNA

a) Physical breakage - pipetting, physical agitation (vortexing), sonication

• Usually leaves blunt ends. 

b) Restriction enzyme digestion

• Cohesive DNA ends for efficient ligation
• Choose enzymes for different average insert sizes
• (e.g. 4-base cutter, 6-base cutter, longer recognition site?)

• May get non-random distribution of fragment sizes, due to non-random distribution of enzyme sites
• Enzymes may not recognise methylated DNA.
• Much eukaryotic DNA is extensively methylated

We can vary time of digestion (or of physical treatment) to produce DNA fragments of desired size.

Partial digestion as a means of isolating longer, overlapping DNA fragments
(Fig 7-13 Lodish et al, 4th ed)

Step 3)     Isolation and ligation of DNA fragments

• Purify fragments of required size by agarose gel electrophoresis, and elution of fragments from gel. Ligate fragments into a vector pre-cut with a restriction enzyme using DNA ligase

Fig 7-8b Lodish et al, 4th ed
 
 

For prokaryotes (smaller genomes) - viable to make gene libraries in plasmids. Plasmid inserts normally ~5-10 kb, so only need a few thousand recombinants for a representative library. For eukaryotes (larger genomes), really need vectors that can hold much larger insert DNA fragments.

Alternative vector systems:
 

	Approximate maximum length of DNA that can be cloned into vectors
Vector type	Cloned DNA (kb)
Plasmid	20
lambda phage	25
Cosmid	45
P1 phage*	100
BAC (bacterial artificial chromosome)	300
YAC (yeast artificial chromosome	1000

Bacteriophage lambda - much more efficient infection of E. coli than achieved by plasmid transformation
*P1- E. coli virus capable of packaging large DNA segments

• In the lytic cycle, the viral DNA is replicated and packaged into > 100 virions per cell. The cell is killed and the virions are released to re-infect more cells.
• In the lysogenic life cycle, the viral DNA integrates into the bacterial chromosome. The DNA is replicated passively as the E. coli cells grow and divide.

Genes involved in lysogeny and certain other genes irrelevant for lytic growth can be deleted from the viral genome, and replaced by other DNA sequences of interest. This is the basis of the use of phage lambda as a vector. Insert size is limited to ~ 25 kb due to the requirement that the DNA has to fit into the phage head.

Fig 7-10b Lodish et al, 4th ed

Making a genomic library in bacteriophage lambda vector:

Fig 7-12 Lodish et al, 4th ed

Phage lambda bind to receptors on the surface of E. coli and injects DNA. Infection by lambda  is much more efficient than plasmid transformation - can get ~ 10⁹ plaques per microgram of DNA, vs ~ 10⁶ colonies per microgram of plasmid DNA

As the bacterial cells lyse, a plaque forms, which spreads as more cells become infected and lyse. The plaque contains the infectious viral particles or virions and each one represents an individual lambda clone (similar to a bacterial colony representing an individual plasmid clone). The appearance is of a clear circle in a 'cloud' of bacterial cells (the bacterial 'lawn'). If incubated for long periods, these circles will continue to grow (containing more and more viral particles) and spread until all bacterial cells are wiped out.

To pick a single lambda  clone, the plaque is 'punched' out from the agar plate and stored in a holding solution. This can then be used to infect more bacterial cells and to replicate more of the recombinant lambda  DNA.

Vectors for making libraries with larger inserts

Cosmid libraries are used for cloning genes with large introns and for sequencing larger chunks of the genome. Cosmid vectors are hybrids of plasmid and bacteriophage lambda DNA (a small ~5 kb plasmid containing the plasmid origin of replication (ori), an antibiotic resistance gene such as amp^r and a suitable restriction site for cloning along with the COS sequence from phage  l DNA). Because of the COS sequence, cosmid recombinants can be packaged into viral particles (allowing high efficiency transformation). Since most of the  l DNA has been discarded, it can be replaced by the DNA of interest, so long as it doesn't exceed the 50 kb limit for packaging into the viral head. The insert sizes are of the order of 35-45 kb. Since the recombinant DNA does not encode any lambda  proteins, cosmids do not form viral particles (or plaques) but rather forms large circular plasmids and the colonies that arise can be selected on antibiotic plates, like other plasmid DNA transformants. Cosmid clones can be manipulated similarly, allowing ease of plasmid isolation (see previous lecture). Since many eukaryotic genes are on the order of 30 - 40 kb, the likelihood of obtaining a DNA clone containing the entire gene sequence is increased significantly when using a cosmid library.

Fig 7-16 Lodish et al, 4th ed

For example, Trp1 mutants can't make tryptophan so can only grow on media supplemented with tryptophan. If the mutant strain is transformed with a YAC containing an intact Trp1 gene, then this will compensate for the inactive gene (complement) and the transfected cell is able to grow on media lacking tryptophan.

YACs are not used as extensively anymore because of inherent problems. For instance, YAC clones can contain non-contiguous segments of the genome. This means that 2 or more DNA fragments from separate parts of the genome can be integrated into an individual YAC (because they are able to support rather large inserts). A second problem is that YACs are unstable and frequently lose parts of the DNA during propagation.

BAC libraries are also used for cloning very large DNA fragments and have been particularly useful for sequencing large genomes. BACs are bacterial artificial chromosomes, and are based on a naturally occurring large bacterial plasmid, the F-factor. BAC vectors can accommodate DNA inserts up to 300 kb, still fairly respectable when needing to clone large genes or map and sequence complex genomes.

BACs have several advantages over YACs, which means they are used more extensively now. Most of the human genome has been sequenced using BAC rather than YAC clones.

Once you have sequenced an organism's genome - what is the next step? Functional genomics

lecture notes last updated 28/8/2002