Cloning the Human MTHFR gene

Initial isolation of the human MTHFR gene was successfully carried out by Goyette et al and reported in 1994.  First, porcine liver MTHFR was isolated using procedures previously outlined (Matthews, 1986).  Following purification of the enzyme, Edmann degradation was performed to determine the amino acid sequence of the protein.  Based upon a 30 amino acid sequence in the enzyme, two degenerate oligonucleotides were designed. From this, a 90bp segment of cDNA was amplified from porcine poly A+ RNA using RT-PCR.  This cDNA sequence was then used to design PCR primers for the PCR screening of a human liver λgt10 cDNA library.  This method proved successful, for a positive clone was detected, sequenced, and verified as MTHFR.  This verification was performed through alignment of the human, porcine, and E. coli genes, which showed numerous areas of sequence similarity. The isolated cDNA clone consisted of 1266bp with one continuous open reading frame.  The fragment coded for the majority of the functional MTHFR gene, although it was not complete at the 5' end (Goyette et al, 1994).   Further work, however, allowed for the isolation of a more complete 2.2kB cDNA sequence, although some information from the 5' region was still lacking (Frosst et al, 1996). 

Using the 2.2kB cDNA, genomic libraries were screened through plaque hybridization, resulting in the isolation of a 17kb genomic DNA fragment for the MTHFR gene.  Analysis of the genomic structure of this fragment indicated that the gene consisted of 11 exons, each of which were 102-432bp in size (Goyette et al, 1998).  Such an analysis, however, gave no information on the gene's regulatory region's structure and details of the 5'end were still incomplete.  However, using 5’-RACE, Homberger et al, (2000) isolated a genomic 4.2kb DNA fragment containing 3.8kb of novel information on the gene’s 5' end.  This discovery completed our knowledge of the MTHFR gene’s functional region and supplied a wealth of information on its regulation (Homberger et al, 2000).