Quickly identify closely related alleles with the CastAway®
Map and Link Human Genetic Disorders with SSLP Analysis
Michelle L. Mack • Douglas J. Wilkin
Medical Genetics Branch, National Human Genome Research Institute National
Institutes of Health, Bethesda, MD
Stratagene’s CastAway ® system is ideal
for analyzing SSLPs (simple sequence-length polymorphisms), or microsatellites.
Different alleles that contain these polymorphisms can vary by just a few bases.
With the CastAway denaturing polyacrylamide gels,* it is now easy to
separate the PCR products containing these polymorphisms. The system
consistently produces high-quality band resolution.
The field of human genetics has greatly accelerated due in part to recent
advances in the development of tools to map human genetic disorders. New methods
generated to discover the chromosomal location of disease genes, and identify
the gene after the map location is known, have aided the rapid identification of
new disease genes. Recognition of the heterogeneity of genetic diseases, the
allelic nature of certain disorders, and mutational heterogeneity in the cause
of genetic conditions may ultimately lead to understanding the relationship
between phenotype and gene.
Simple Sequence-Length Polymorphism Analysis
To identify disease loci, small fragments of genomic DNA must be analyzed.
These markers, SSLPs or microsatellites, are derived from unique stretches of
DNA that contain very short, simple-sequence repeats.1 Each
microsatellite marker is made up of a variable number of di-, tri-, or
tetranucleotide repeats at a particular location.1 Often these
markers are (CA)n repeat polymorphisms.1 These polymorphisms can
easily be genotyped by PCR with primers that anneal to single-copy DNA, which
flanks the repetitive element.
Genotyping, using microsatellite markers, at specific locations throughout
the genome allows inheritance patterns of specific pieces of chromosomes within
a family to be determined. Haplotypes are generated by analyzing individual
members of a pedigree with a number of closely linked markers. These haplotypes
allow the transmission of a particular locus through a family to be analyzed;
furthermore, they determine whether that locus is linked to a particular
phenotype, as well as aid in identifying recombinational events, which define a
critical region for a disease locus. Statistical programs, such as FASTLINK,2,3
are then employed to determine if a particular haplotype occurs only with the
disease state; if a statistically significant correlation can be made between a
particular marker at a given location and a particular disease, then the disease
is linked to this marker at a known location within the genome.
Use the CastAway® System for Mapping Studies
Employing polymorphic markers in a mapping study relies upon the method used
to separate the particular bands generated by PCR. In a novel approach, we used
CastAway precast polyacrylamide gels instead of pouring our own gels. The latter
traditional method is not only tedious and labor intensive but is also open to
problems such as bubble formation and leaking. Because CastAway gels are
precast, they eliminate the time needed to prepare traditional polyacrylamide
gels. These prepoured gels are available with or without preformed wells. In
addition, the thinner gel format (0.25 mm), as well as the various options of
polyacrylamide compositions, allowed us to effectively resolve PCR products,
which often ranged in size from 120 bp to 400 bp and sometimes differed by only
a few base pairs. The CastAway system permitted us to generate reproducible
data; hence, variability between the gels, which may affect band resolution, was
minimized. As a result, our studies, which easily could have taken many months,
were completed in a just a few days or weeks.
Linkage Studies Analyzed
We used Stratagene’s CastAway system to analyze linkage studies of many
families with inherited disorders. Stickler syndrome is one of the milder
phenotypes that results from mutations in the gene encoding type II collagen,
COL2A1.4,5 Genetic heterogeneity has been demonstrated for Stickler
syndrome, with mutations in two genes that encode type XI collagen, COL11A1 and
COL11A2, and in at least one additional unidentified gene, resulting in Stickler
syndrome.6,7 To determine if the phenotype in a family with Stickler
syndrome is linked to the COL11A1 or COL11A2 locus (COL2A1 was previously
excluded as the Stickler syndrome gene in this family7), polymorphic
markers were analyzed by PCR amplification of the genomic DNA. A (CA)n repeat
microsatellite marker, D1S206, was analyzed as one of a number of polymorphic
markers in chromosome 1p21. The markers were analyzed to generate haplotypes
across the region of chromosome 1 containing COL11A1. Analysis of polymorphic
markers within the COL11A1 gene and a-amylase (AMY2B)
gene completed the haplotypes. Analysis of these haplotypes excluded COL11A1 as
a candidate for Stickler syndrome in this pedigree. Haplotype analysis, using
markers in chromosome 6p21.3, also excluded COL11A2 as a candidate for Stickler
syndrome in this pedigree.
In a genotyping experiment using the marker D1S206, PCR products were analyzed
on a denaturing polyacrylamide gel to resolve allelic differences that
were greater than 2 bp (Figure
1, Panel A). Five alleles, labeled A through E (Figure
1, Panel A) were identified for this marker, with each allele differing
by at least 2 bp, representing multiples of (CA) dinucleotides. Each allele
is represented by multiple bands (“stutter” bands), which may
be produced from Taq DNA polymerase slippage during the amplification
process. These bands resemble a 2-bp ladder, with the most common band
often being 2 bp shorter than a major band. Artifact bands can be distinguished
from real bands by identifying a heterozygous individual, with two sets
of multiple bands. We observed heterozygous patterns (Figure
1, Panel A, Lanes 1, 3-7, 9, and 13-17), 2-bp differences between
the upper alleles (Figure
1, Panel A, Lane 6 compared to Lane 7 and Lane 13 compared to Lane
14), and homozygous individuals with only one set of bands (Figure
1, Panel B, Lanes 3, 6, 8, 11, 12, 15, and 16). For each marker, the
dominant band is designated the “true” allele. Because the pattern
of stutter bands is usually consistent for each marker, the genotype of
each individual is deduced by scoring the dominant bands only.
We also used the CastAway system to analyze linkage studies of families with
cartilage hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia
characterized by dwarfism, decreased immunity, and fine, sparse hair.8
CHH was previously linked to chromosome 9p13.9 D9S165 was analyzed
as one of a number of markers in chromosome 9p13 to further define the
segment of chromosome 9p13 to which the phenotype is linked. In a genotyping
experiment using the marker D9S165, three alleles, labeled A through C
1, Panel B), were identified. Additional examples of heterozygous
patterns were observed in Figure
1, Panel B, Lanes 1, 4, 5, 7, 10, 17, and 20.
By analyzing this marker and others using the CastAway system, haplotypes
were generated, which further defined a critical region of chromosome 9 linked
to CHH in this pedigree (not shown). Experiments to identify the CHH gene
Use the CastAway system as a convenient, time-saving alternative to
traditional poured polyacrylamide gels for mapping studies. Because linkage
mapping relies upon a reproducible resolution of bands, which are only minutely
different in size, the CastAway system greatly increases the likelihood of
correctly identifying alleles and provides more time to test additional markers.
Polymorphic markers were analyzed by PCR amplification of genomic DNA in the
presence of a-[35S]dATP, followed by analysis on
CastAway 4.5% or 6% denaturing polyacrylamide gels. PCR amplifications were
performed in 25-µl reactions containing 50-ng of genomic DNA; 2 units of Taq
DNA polymerase; 2.5 pMol of each primer; 200 µM of dCTP, dTTP, and dGTP; 2.5
µM dATP; 2 mM MgCl2; 10 mM Tris-HCl pH 8.3; 50 mM KCl; and 2 µCi of a-[35S]dATP.
Amplification conditions consisted of an initial incubation of 2 minutes at
94ºC followed by 35 cycles of 94ºC for 1 minute, 60ºC for 1 minute, and 72ºC
for 1 minute followed by a 9-minute incubation at 72ºC. Oligonucleotide primers
were purchased from Research Genetics.
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* U.S. Patent No. 5,837,288