Rapid detection of genomic,,,duplications and deletions using,,,the Agilent 2100 bioanalyzer
but has proved technically difficult
in practice. The fact that each
primer pair has to be optimized
when combined in a given PCR reaction limits the mix and
match potential. FISH is a visual
method, meaning that the number
of signals seen corresponds to the
copy number of the probe. The
disadvantages are the time-consuming
before the analysis can be performed
and the relatively low resolution
(approx. 40 kb for a cosmid
probe). Although this is more than
adequate for larger rearrangements,
small (e.g. single exon) deletions
and duplications are not usually
detected with this method. Two
high resolution, high throughput
techniques have recently been
described for quantitative analysis
of DNA, namely multiplex amplifiable
probe hybridization (MAPH)1
and multiplex ligation-dependent
probe amplification (MLPA)2. Both
are based on quantitative amplification
using a single pair of
primers following probe hybridization
to genomic DNA. The difference
lies in the probes and the
way the genomic DNA is handled.
MAPH probes are PCR products
that are hybridized to genomic
DNA immobilized onto a nylon
membrane. After hybridization the
unbound probes are removed by
stringent washing, with the bound
probes being subsequently denatured
and amplified in a PCR reaction.
As the probes are designed
to all have identical sequences at
each end, the PCR reaction can
be performed with a single primer
pair. MLPA is a ligation-based
assay, with 2 single stranded DNA
sequences being ligated together
only if they both hybridize adjacently
to their complementary
sequences on genomic DNA. As
unligated probes can not be PCR
amplified it is not necessary to
immobilize the genomic DNA (in
contrast to MAPH), and the washing
steps can also be skipped.
Different methods for the analysis
and the quantitation of the PCR
products have been describe
Source:Page: All 1 2 3 4 5 Related biology technology :1
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