Gene Cloning

14 Gene Cloning
Box 2.1 Simple Sequence Repeats (SSRs)
Simple sequence repeats (SSRs) are found in all eukaryotes, although their
relative abundance does vary between organisms. When genomic DNA is
isolated by centrifugation on a cesium chloride gradient it forms a discrete
band although smaller satellite bands are often visible. These contain simple
sequence repeat DNA which is also known as satellite DNA. SSRs can
be classified into three categories, satellite DNA, mini-satellite DNA and
micro-satellite DNA.
Satellite DNA sequences are typically greater than 100 kb in length and
composed of repeats five to 200 bp in length. An example is the alphoid
DNA found in centromeric DNA, in which a 171 bp sequence is repeated
many times, resulting in a region of around 500 kb.
Mini-satellite sequences are generally less than 20 kb of DNA composed of
repeats less than 25 bp in length. An example of a mini-satellite sequence
is one found near the insulin gene, which has the sequence
5′-ACAGGGGTGTGGGG-3′. The number of repeats of the mini-satellite
sequence at each locus varies between individuals and they are therefore
also referred to as variable number tandem repeats or VNTRs. It was analysis
of these VNTRs that formed the original basis of DNA profiling.
Micro-satellite DNA is normally less than 150 bp in length and composed
of 5 bp repeat sequences. Micro-satellites are also known as simple tandem
repeats (STRs). An example is the D18S51 micro-satellite, which has
the sequence 5′-AGAA-3′. Most individuals will have between seven and 27
copies of D18S51 on each copy of chromosome 18. Analysis of these simple
tandem repeats has formed the basis of modern DNA profiling techniques
(Section 13.2).
accumulation of these sequences in eukaryotic genomes over evolutionary
time. Similarly, over time these transposons have been subject to mutations
and deletions, resulting in most cases in the loss of the ability to
transpose. There are four classes of transposable elements, namely: LTR
retroposons (retrovirus-like elements), long interspersed elements
(LINEs), short interspersed elements (SINEs) and DNA transposons; a
more detailed discussion of interspersed transposon-derived repeats can
be found in Box 2.2.
In addition to the multiple repeat elements mentioned above, approximately
5% of the genome appears to be a duplication of another segment
of the genome. There are several mechanisms that, during evolution, result
in sections of the genome being copied and pasted into the same or different
location on the chromosome. Often the duplicated segment will have