IST Scholarship Proposal - Example

 

 

Introduction:  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.

  

Methods:  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 detected.

 

Objectives:

1) 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 method.

 

Significance and uniqueness of the project: APEX 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

 

Specific outcomes of the project tasks

 

References

  1. 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
  2. Jacques, P.F. et al. (2001). Determinants of plasma total homocysteine concentration in the Framingham Offspring cohort. Am J Clin Nutr; 73:613-621.
  1. Chappell, Jean et al. Correlation between MTHFR677(C>T), Hyperhomocysteinemia and BMI  Poster presented at American Society of Clinical Laboratory Sciences conference, July 2003
  2. 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.
  3. 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.

 

Budget

 

Student Stipend:   $1000