“Different types of markers used to construct genetic maps and
scoring”
Mapped features that are not genes are called DNA markers. As with gene
markers, a DNA marker must have at least two alleles to be useful. There
are three types of DNA sequence features that satisfy this requirement:
➔ Restriction fragment length polymorphisms (RFLPs).
➔ Simple sequence length polymorphisms (SSLPs).
➔ Single nucleotide polymorphisms (SNPs).
1. Restriction fragment length polymorphisms (RFLPs):
● RFLPs were the first type of DNA marker to be studied.
● The sequence specificity means that treatment of a DNA molecule
with a restriction enzyme should always produce the same set of
fragments.
● This is not always the case with genomic DNA molecules because
some restriction sites are polymorphic, existing as two alleles, one
allele displaying the correct sequence for the restriction site and
therefore being cut when the DNA is treated with the enzyme, and the
second allele having a sequence alteration so the restriction site is no
longer recognized.
● The result of the sequence alteration is that the two adjacent
restriction fragments remain linked together after treatment with the
enzyme, leading to a length polymorphism.
● The RFLP and its position on a genome map can be worked out by
following the inheritance of its alleles, just as is done when genes are
used as markers.
● There are thought to be about 105 RFLPs in the human genome, but
of course for each RFLP there can only be two alleles (with and
without the site).
● The value of RFLPs in human gene mapping is therefore limited by
the high possibility that the RFLP being studied shows no variability
among the members of an interesting family.
, Scoring:
● In order to score an RFLP, it is necessary to determine the size of just
one or two individual restriction fragments against a background of
many irrelevant fragments.
● This is not a trivial problem: an enzyme such as EcoRI, with a 6bp
recognition sequence, should cut approximately once every 46 =
4096 bp and so would give almost 800 000 fragments when used
with human DNA.
● After separation by agarose gel electrophoresis, these 800 000
fragments produce a smear and the RFLP cannot be distinguished.
● Southern hybridization, using a probe that spans the polymorphic
restriction site, provides one way of visualizing the RFLP , but
nowadays PCR is more frequently used.
● The primers for the PCR are designed so that they anneal either side
of the polymorphic site, and the RFLP is typed by treating the
amplified fragment with the restriction enzyme and then running a
sample in an agarose gel.
2. Simple sequence length polymorphisms (SSLPs):
SSLPs are arrays of repeat sequences that display length variations,
different alleles containing different numbers of repeat units. Unlike RFLPs,
SSLPs can be multi-allelic as each SSLP can have a number of different
length variants. There are two types of SSLP:
scoring”
Mapped features that are not genes are called DNA markers. As with gene
markers, a DNA marker must have at least two alleles to be useful. There
are three types of DNA sequence features that satisfy this requirement:
➔ Restriction fragment length polymorphisms (RFLPs).
➔ Simple sequence length polymorphisms (SSLPs).
➔ Single nucleotide polymorphisms (SNPs).
1. Restriction fragment length polymorphisms (RFLPs):
● RFLPs were the first type of DNA marker to be studied.
● The sequence specificity means that treatment of a DNA molecule
with a restriction enzyme should always produce the same set of
fragments.
● This is not always the case with genomic DNA molecules because
some restriction sites are polymorphic, existing as two alleles, one
allele displaying the correct sequence for the restriction site and
therefore being cut when the DNA is treated with the enzyme, and the
second allele having a sequence alteration so the restriction site is no
longer recognized.
● The result of the sequence alteration is that the two adjacent
restriction fragments remain linked together after treatment with the
enzyme, leading to a length polymorphism.
● The RFLP and its position on a genome map can be worked out by
following the inheritance of its alleles, just as is done when genes are
used as markers.
● There are thought to be about 105 RFLPs in the human genome, but
of course for each RFLP there can only be two alleles (with and
without the site).
● The value of RFLPs in human gene mapping is therefore limited by
the high possibility that the RFLP being studied shows no variability
among the members of an interesting family.
, Scoring:
● In order to score an RFLP, it is necessary to determine the size of just
one or two individual restriction fragments against a background of
many irrelevant fragments.
● This is not a trivial problem: an enzyme such as EcoRI, with a 6bp
recognition sequence, should cut approximately once every 46 =
4096 bp and so would give almost 800 000 fragments when used
with human DNA.
● After separation by agarose gel electrophoresis, these 800 000
fragments produce a smear and the RFLP cannot be distinguished.
● Southern hybridization, using a probe that spans the polymorphic
restriction site, provides one way of visualizing the RFLP , but
nowadays PCR is more frequently used.
● The primers for the PCR are designed so that they anneal either side
of the polymorphic site, and the RFLP is typed by treating the
amplified fragment with the restriction enzyme and then running a
sample in an agarose gel.
2. Simple sequence length polymorphisms (SSLPs):
SSLPs are arrays of repeat sequences that display length variations,
different alleles containing different numbers of repeat units. Unlike RFLPs,
SSLPs can be multi-allelic as each SSLP can have a number of different
length variants. There are two types of SSLP:
