Human BAC library

Construction and rapid screening

Shuichi Asakawa, Izumi Abe, Yoshiki Kudoh, Noriyuki Kishi, Yimin Wang, Ryo Kubota, Jun Kudo, Kazuhiko Kawasaki, Shinsei Minoshima, Nobuyoshi Shimizu

Research output: Contribution to journalArticle

152 Citations (Scopus)

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 languageEnglish
Pages (from-to)69-79
Number of pages11
JournalGene
Volume191
Issue number1
DOIs
Publication statusPublished - 1997 May 20

Fingerprint

Human Artificial Chromosomes
Bacterial Artificial Chromosomes
Clone Cells
DNA
Polymerase Chain Reaction
B-Lymphoid Precursor Cells
Chromosome Walking
Human Genome
Southern Blotting
Diploidy
Karyotype
Libraries

Keywords

  • Bacterial artificial chromosome
  • Four-dimensional PCR
  • Human genomic library
  • Vectorette

ASJC Scopus subject areas

  • Genetics

Cite this

Asakawa, S., Abe, I., Kudoh, Y., Kishi, N., Wang, Y., Kubota, R., ... Shimizu, N. (1997). Human BAC library: Construction and rapid screening. Gene, 191(1), 69-79. https://doi.org/10.1016/S0378-1119(97)00044-9

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 journalArticle

Asakawa, S, Abe, I, Kudoh, Y, Kishi, N, Wang, Y, Kubota, R, Kudo, J, Kawasaki, K, Minoshima, S & Shimizu, N 1997, 'Human BAC library: Construction and rapid screening', Gene, vol. 191, no. 1, pp. 69-79. https://doi.org/10.1016/S0378-1119(97)00044-9
Asakawa S, Abe I, Kudoh Y, Kishi N, Wang Y, Kubota R et al. Human BAC library: Construction and rapid screening. Gene. 1997 May 20;191(1):69-79. https://doi.org/10.1016/S0378-1119(97)00044-9
Asakawa, Shuichi ; Abe, Izumi ; Kudoh, Yoshiki ; Kishi, Noriyuki ; Wang, Yimin ; Kubota, Ryo ; Kudo, Jun ; Kawasaki, Kazuhiko ; Minoshima, Shinsei ; Shimizu, Nobuyoshi. / Human BAC library : Construction and rapid screening. In: Gene. 1997 ; Vol. 191, No. 1. pp. 69-79.
@article{09012386ebd3495191cc97ecde9aa797,
title = "Human BAC library: Construction and rapid screening",
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.",
keywords = "Bacterial artificial chromosome, Four-dimensional PCR, Human genomic library, Vectorette",
author = "Shuichi Asakawa and Izumi Abe and Yoshiki Kudoh and Noriyuki Kishi and Yimin Wang and Ryo Kubota and Jun Kudo and Kazuhiko Kawasaki and Shinsei Minoshima and Nobuyoshi Shimizu",
year = "1997",
month = "5",
day = "20",
doi = "10.1016/S0378-1119(97)00044-9",
language = "English",
volume = "191",
pages = "69--79",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1",

}

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 -