Segments missing from the draft human genome sequence can be isolated by transformation-associated recombination cloning in yeast.
The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were re-isolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E. coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.
Kouprina
Natalay
N
Laboratory of Biosystems and Cancer, National Cancer Institute, Building 37, Room 5031, Bethesda, Maryland 20892, USA. kouprinn@mail.nih.gov
Leem
Sun-Hee
SH
Solomon
Greg
G
Ly
Albert
A
Koriabine
Maxim
M
Otstot
John
J
Pak
Eugene
E
Dutra
Amalia
A
Zhao
Shaying
S
Barrett
J Carl
JC
Larionov
Vladimir
V
D015815:Cell Adhesion Molecules / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins; D015894:Genome, Human; D016045:Human Genome Project; D006801:Humans; D017436:Kallmann Syndrome / Q000235:genetics; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
C076266:ANOS1 protein, human; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D004247:DNA