Brugia Malayi - Clark Science Center - Smith College

What’s Your Orientation: Long PCR Analysis of the Xq28 Region
Cait Kirby
On the XQ28 region of the X chromosome there are two genes flanked by two repeat regions. The orientation of these genes and
repeat regions is variable, with 33% of females presenting as heterozygous. (Small et al) The two genes that exist in this region,
emerin and filamin, serve to produce muscle and structural tissue. If any part of the emerin gene is deleted a patient could have
muscular dystrophy; if any part of the filamin gene is deleted, the embryo is inviable. Emerin, 2kb in length, and filamin, 26kb long,
are sandwiched between two 11.3kb repeat regions which have 99% sequence identity (Small et al). Inversions inhibit recombination
via less frequent crossing over, which contributes to speciation (Small et al).
The goal of this project is to design a teaching lab to provide students with a hands-on learning opportunity to understand
concepts such as crossing over, recombination, and linkage disequilibrium. This teaching lab will also give students an opportunity
to delve into lab work, including DNA extraction, PCR, and gel electrophoresis. In designing this experiment I used information
from scientists at Emory University who provided us with primer sequences, and a PCR program. After determining novel DNA
extraction methods, the PCR began to work, though inconsistently. After deciding to add DMSO (to improve denaturation of G-C
rich regions) and BSA (to bind up inhibitors) both of which are purported to enhance the efficiency of PCR, we became successful,
though still rather inconsistently.
The goal of this project is to produce a presence-absence assay allowing us to quickly and easily determine in which orientation
a student’s genes lie. The primers that we were provided produce two different sized bands, the forward orientation results in an
11kb band, while the inverted orientation results in a 12kb band. The difference is slight, and typically not discernible, but I will still
attempt to multiplex the PCR – putting both assays into one reaction – providing us with one quick answer instead of two separate
reactions. (Supported by the Nancy Kay Holmes Fund)
Advisor: Robert Merritt
References:
Small, Iber & Warren. Emerin deletion reveals a common X-chromosome inversion mediated by inverted repeats. Nat Genet. 1997 May ;16(1):96-9.
2012
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