Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products.
An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.
Moir
D T
DT
Department of Human Genetics and Molecular Biology, Collaborative Research, Inc., Waltham, MA 02154.
Dorman
T E
TE
Xue
F
F
Ma
N S
NS
Stanton
V P
VP
Housman
D
D
Bowden
D W
DW
Noll
W W
WW
Mao
J
J
D001483:Base Sequence; D016386:Chromosome Walking; D018244:Chromosomes, Artificial, Yeast; D002879:Chromosomes, Human, Pair 10; D017931:DNA Primers; D008040:Genetic Linkage; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D009377:Multiple Endocrine Neoplasia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping
D017931:DNA Primers; D005819:Genetic Markers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas