Scholarship Proposal - Example
Numerous studies have found a linkage between MTHFR
(5,10-methylenetetrahydrofolate reductase) 677 and 1298 gene variants and
elevated homocysteine levels and cardiovascular disease (2). Our previous work
used restriction fragment length polymorphisms of these two versions of the
MTHFR gene to genotype 577 individuals with a family history of obesity (1,3).
The assumption is that a single nucleotide change resulting in an amino acid
substitution has occurred. However, this method can be nonspecific, since the
loss of the restriction enzyme site can result from a mutation in any one of
four nucleotides in the recognition site. DNA sequencing can also differentiate
between all the 16 possible sequences. My proposal is to reexamine the already
genotyped samples using a newly developed rapid assay for human identity typing
based on arrayed primer extension (APEX) analysis (4,5). The APEX results will
be confirmed by Pyrosequencing by Dr. Mark Flood using the materials I prepare.
DNA Purification was performed using QIAamp spin columns. PCR amplification was
performed using a forward 5’amino modified end primer and a reverse 5’
biotinylated end primer (6). Microcon filters are used to purify the PCR
products from unincorporated oligonucleotides. Magnetic streptavidin beads are
used to separate the amino modified and biotinylated strands. The
amino-modified strand is used for the array and the biotin strand is used for
Pyrosequencing. The amino-modified PCR product is hybridized to the sequencing
extension primers (these also have a 5’ amino modified end) located 1 base from
the variant nucleotide. They are then spotted onto ArrayIt SuperAldehyde slides
using the Spotbot microarrayer. After spotting, the slides will be taken to the
microarray facility, where the PCR products can be hybridized, the single base
extension primers can be extended on the arrays, and the signals can be
Develop a new procedure for genotyping MTHFR 677 and MTHFR 1298 using arrayed
primer extension (APEX) analysis. 2) Confirm the genotypes of 577 individuals
genotyped previously with RFLP genotyping using Pyrosequencing and the new
Significance and uniqueness
of the project:
analysis promises to be a powerful means for automating SNP genotyping by
eliminating the need for the labor-intensive step of gel electrophoresis. It is
a significant improvement over DNA sequencing which requires one reaction per
well in plate assays. APEX arrays can be expanded to type very large numbers of
SNPs simultaneously. MTHFR genotyping using APEX is an ideal technique to
develop, since the process can be expanded. West Virginia can be an especially
good place to expand this technique due to the high frequency of MTHFR mutations
and cardiovascular disease in its population.
Approach used to
achieve the objectives
Spot and hybridize a
subset of PCR samples onto the test array.
Extend primers on the
Visualize the results.
Modify to get the best
Perform APEX on the
remainder of samples.
Compare results to the
genotypes obtained using RFLP.
Specific outcomes of
the project tasks
Develop a new procedure
to determine MTHFR 677 and 1298 genotypes.
Confirm the MTHFR 677
and 1298 genotypes of 520 individuals.
Present results at
Sigma Xi Research Day and seek to publish findings.
Jeong, Jooha et al.
Homocysteine and MTHFR Genotype in an Obese West Virginia Population
Poster presented at MU Sigma Xi Research Day, April 25, 2003
et al. (2001).
Determinants of plasma total homocysteine concentration in the Framingham
Offspring cohort. Am J Clin Nutr; 73:613-621.
Chappell, Jean et al.
Correlation between MTHFR677(C>T), Hyperhomocysteinemia and BMI
Poster presented at American Society of Clinical Laboratory Sciences
conference, July 2003
Landi S, Gemignani F,
Gioia-Patricola L, Chabrier A, Canzian F. Evaluation of a microarray for
genotyping polymorphisms related to xenobiotic metabolism and DNA repair.
Biotechniques. 2003 Oct; 35(4):816-820.
Jaakson K, Zernant J,
Kulm M, Hutchinson A, Tonisson N, Glavac D, Ravnik-Glavac M, Hawlina M,
Meltzer MR, Caruso RC, Testa F, Maugeri A, Hoyng CB, Gouras P, Simonelli F,
Lewis RA, Lupski JR, Cremers FP, Allikmets R. Genotyping microarray (gene
chip) for the ABCR (ABCA4) gene. Hum Mutat. 2003 Nov; 22(5):395-403.
6. Frosst P, Blom HJ,
Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ,den Heijer M,
Kluijtmans LA, van den Heuvel LP. A candidate genetic risk factor for vascular
disease: a common mutation in methylenetetrahydrofolate reductase. Nat
Genet. 1995 May; 10(1):111-3.