Abstract
We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.
| Original language | English |
|---|---|
| Pages (from-to) | 69-79 |
| Number of pages | 11 |
| Journal | Gene |
| Volume | 191 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1997 May 20 |
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Keywords
- Bacterial artificial chromosome
- Four-dimensional PCR
- Human genomic library
- Vectorette
ASJC Scopus subject areas
- Genetics
Cite this
Human BAC library : Construction and rapid screening. / Asakawa, Shuichi; Abe, Izumi; Kudoh, Yoshiki; Kishi, Noriyuki; Wang, Yimin; Kubota, Ryo; Kudo, Jun; Kawasaki, Kazuhiko; Minoshima, Shinsei; Shimizu, Nobuyoshi.
In: Gene, Vol. 191, No. 1, 20.05.1997, p. 69-79.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Human BAC library
T2 - Construction and rapid screening
AU - Asakawa, Shuichi
AU - Abe, Izumi
AU - Kudoh, Yoshiki
AU - Kishi, Noriyuki
AU - Wang, Yimin
AU - Kubota, Ryo
AU - Kudo, Jun
AU - Kawasaki, Kazuhiko
AU - Minoshima, Shinsei
AU - Shimizu, Nobuyoshi
PY - 1997/5/20
Y1 - 1997/5/20
N2 - We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.
AB - We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.
KW - Bacterial artificial chromosome
KW - Four-dimensional PCR
KW - Human genomic library
KW - Vectorette
UR - http://www.scopus.com/inward/record.url?scp=0030980842&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030980842&partnerID=8YFLogxK
U2 - 10.1016/S0378-1119(97)00044-9
DO - 10.1016/S0378-1119(97)00044-9
M3 - Article
VL - 191
SP - 69
EP - 79
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -