A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

TY - JOUR

T1 - A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

AU - She, Qunxin

AU - Confalonieri, F.

AU - Zivanovic, Y.

AU - Medina, N.

AU - Billault, A.

AU - Awayez, Mariana J.

AU - Thingoc, Hoa Phan

AU - Thi Pham, Boi-Tien

AU - van der Oost, J.

AU - Duguet, M.

AU - Garrett, Roger A.

N1 - Keywords: Sulfolobus; genome mapping; paired-PCR; BAC clones

PY - 2000

Y1 - 2000

N2 - The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR screening. The PCR approaches included a novel chromosome walking method termed “paired-PCR”. 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half the genome sequence has been accumulated.

AB - The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR screening. The PCR approaches included a novel chromosome walking method termed “paired-PCR”. 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half the genome sequence has been accumulated.

U2 - 10.3109/10425170009033231

DO - 10.3109/10425170009033231

M3 - Journal article

SN - 1940-1736

VL - 11

SP - 183

EP - 192

JO - Mitochondrial DNA

JF - Mitochondrial DNA

IS - 3 & 4

ER -