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al., 2009, with capability of reading 400 K—4 M sequences compared with the traditional 96 capillary, and reading length from 25 to 450 basepairs, depending
on the platform Mardis, 2008. In shotgun sequencing, the genome is cut up into smaller fragments of DNA which can be massively sequenced in parallel.
Subsequently, the sequenced fragments are assembled into contigs based on the overlap in the sequence reads or, alternatively, aligned and compared to a
reference genome which will bring to disease-gene identification. This promising method, however, still limited by its high cost. Clinical and biological
interpretation the variants resulted from this method will require large international and multidisciplinary collaborative efforts Visser et al., 2009
II.3. X-Chromosome Linkage Analysis
Before researchers could elucidate and finally sequence the gene responsible for a disease, it must be first mapped, located in the Genome. Genetic
linkage analysis plays role in identification regions of the genome containing genes locus that predispose to disease by use of observations of related
individuals Teare and Barret, 2005. This method works using short tandem repeat STR-markers or microsatellite, a well-characterized regions of DNA that
consist of multiple repeats of a short sequence typically 2–8 bp and highly show genetic variation polymorphism in nature Weber, 1990. Researcher are looking
for a marker that is consistently present in those that are affected, and is not present in non-affected relatives, assuming that a causative genetic variant is
likely to lie close to that marker Burton et al. 2005. Linkage analysis work on the principle of cosegregation of stretches of DNA in families rearranged by
recombination events in meiosis. The probability of recombination between two loci at meiosis is called recombination fraction , which can be utilized as a
stochastic measure for the genetic distance between two genes Massanet, 2009. The further apart two loci are from each other on a chromosome, the greater the
probability is that a recombination will occur between themhypothesis null assumes no linkage, or
=0.5 Teare and Barret, 2005. Two loci segregate together more often if they are located close enough together on the same
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chromosome in other words the chance of recombination is less than 50, or alternative hypothesis assumes linkage exists 12 Burton et al. 2005. The
expected numbers of recombination occurring between two loci on a single chromatid during meiosis is called genetic map distance in units of Morgans
Teare and Barret, 2005. Linkage is described in linkage interval and scored in logarithm of the odds LOD score, a function of the recombination which
indicates how much higher the likelihood of the data is under linkage than under the absence of linkage Massanet, 2009.
2 1
log
10
= =
θ θ
θ likelihood
likelihood LOD
Morton 1955 proposed a critical value of LOD score=2 for significant linkage in X-linked locus Morton, 1955. Linkage can be excluded from the region if the
LOD score is below -2. This approach is called exclusion mapping Massanet, 2009. In mental retardation, linkage analysis is often used as first stage to narrow
down region of interest into linkage interval in effort to find evidence of containing a disease gene Teare and Barret, 2005. Candidate gene present in the
linkage interval can be used as a target of sequencing to find the disease causing genes Lugtenberg et al., 2006.
II.4. X-Chromosome Inactivation
