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,
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
(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).