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Lambda DNA

Bioinformatics
Sequence notation
Lambda DNA
pGT4
Restriction enzymes
pGTλclones
Sequences
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About Lambda DNA

The sequence of bacteriophage Lambda DNA.
Lambda digests gel

About samples containing Lambda-DNA:
Lambda is a medium size E.coli bacteriophage. The DNA molecule of 48502 basepairs is linear and except for the extreme ends double-stranded. At each end the 5' strand overhangs the 3' strand by 12 bases. The sequences of the ends are complementary. At ambient temperatures, in a solution containing purified Lambda-DNA these so-called 'cos ends' may pair and form the so-called 'cos-site'. As a consequence, the DNA is (partly) circularised or have formed concatemers.
In a purified- DNA solution the cos-site can be destroyed by heating 10 minutes at 65-70 C. Immediate cooling in ice-water prevents reformation of the "cos-site".

note:
The 12 nucleotide 5 overhangs at the cos-ends of the linear Lambda DNA are the result of a cut by the enzyme terminase. This enzyme is encoded by Lambda itself and acts like a restriction enzyme during the replication of the phage DNA. It is an endonuclease specific for the cos-site in multimeric phage DNA. The ends of the resulting monomeric DNA (called cos ends) are similar to the sticky (or cohesive) ends produced by common restriction enzymes. Because they have long 5 overhangs (12 nucleotides) cos ends are much more sticky than the cohesive ends generated by restriction enzymes which have generally an overhang of 4 or 2 nucleotides.

Since cos ends have complementary overhangs they are compatible for efficient ligation.

The ends in detail:
 

 

After electrophoresis, the gel band pattern of a Lambda DNA digest may show an extra band, due to the joining (and forming the cos-site) of the two end fragments. If this happens (it will always, to some extent..), the bands containing the separate end fragments will be present in an amount lower than calculated..

Example:

   
The gel on the left shows the gel electrophoresis result
of two BstE II digests of Lambda DNA.
Before pipetting the samples into the slots, the digest for lane 2 was heated for 10 minutes at 70C, to destroy the cos-site.

When the digest is not heated before the gel run (lane 1), a number of b and c fragments have joined to form band a, which is in size the sum of b and c.
It is very likely that a number of the Lambda DNA molecules was circular (because of the cos-site formed) already before the digestion.

When you use EMBOSS>Restrict to find out about the fragment sizes, you will not find band a. You will also find out, that b and c are end fragments.
When you use EMBOSS>restrict to "do" the BstE II digestion with the option Allow circular DNA? > YES, you will not find fragments b and c in the output list, but fragment a instead.
When you use ApE to find the BstE II fragment lengths, it will depend on state of the linear/circular button (top right in the ApE window) whether you'll find a, or  b and c.

note:
Restriction enzymes often generate "cohesive" or "sticky" ends. Those are ends with short, mostly 4 nucleotides long, single-stranded overhangs. Because the overhangs are much shorter than the Lambda cos-ends, annealing of those ends is much less stable. After gel electrophoresis, you'll never observe additional DNA bands, formed by fragments joined by restriction enzyme generated "sticky" ends.

 

Map

This is a BamHI-EcoRI restriction map of Lambda DNA:

(read it like this: (e.g.) "the 4878 bp long EcoRI fragment is between the first and the second EcoRI site, and in the Simple Cloning Lab it is called Eco E" ).

The staggered ends of the black bar indicate the single-stranded cos ends of the Lambda DNA, which can anneal to form the cos site (BamC, BamF, EcoA and EcoF are end fragments).
The exact positions of the BamHI and EcoRI (and other) restriction sites can be found in the table below.

 

Restriction sites list

An output file from EMBOSS>restrict for restriction enzyme sites in Lambda DNA can be found here. The list shows all enzymes with a 6 basepairs recognition site ("6cutters") in alphabetical order.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supervisor Neil Johnson's Lambda DNA digests gel