Identification of human chromosome 9 specific genes using exon amplification.
We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA.
Church
D M
DM
Molecular Neurogenetics Unit, Massachusetts General Hospital, Boston.
Banks
L T
LT
Rogers
A C
AC
Graw
S L
SL
Housman
D E
DE
Gusella
J F
JF
Buckler
A J
AJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping; D002899:Chromosomes, Human, Pair 9; D003360:Cosmids; D017931:DNA Primers; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D012313:RNA