GENOMICS 34, 213–218 (1996)
ARTICLE NO. 0268
Construction and Characterization of a Human Bacterial
Artificial Chromosome Library
UNG-JIN KIM, BRUCE W. BIRREN, TATIANA SLEPAK, VALERIA MANCINO, CECILIE BOYSEN,
HYUNG-LYUN KANG, MELVIN I. SIMON, AND HIROAKI SHIZUYA1
Division of Biology, 147-75, California Institute of Technology, Pasadena, California 91125
Received January 3, 1996; accepted March 28, 1996
We have constructed an arrayed human genomic
BAC library with approximately 41 coverage that is
represented by 96,000 BAC clones with average insert
size of nearly 140 kb. A new BAC vector that allows
color-based positive screening to identify trans-
formants with inserts has increased BAC cloning ef-
ficiency. The library was gridded onto hybridization
filters at high density for efficient identification of
BAC clones by colony hybridization. The library was
also formulated into characteristic DNA pools to
allow for PCR screening of the library for STS con-
tent. We have characterized the library mainly by
screening with more than 300 different landmarks
that include cDNA, STSs, and cosmid clones. We de-
scribe methods for using BAC clones and discuss the
implications for genome characterization, mapping,
and sequencing. q 1996 Academic Press, Inc.
INTRODUCTION
Mapping and analysis of complex genomes requires
application of a variety of resources and techniques.
For example, YAC libraries containing human DNA
inserts up to 2 Mb have been essential in building
frameworks for genome-wide physical mapping (Chu-
makov et al., 1995; Hudson et al., 1995). In addition,
for some human chromosomes, YAC-based maps that
incorporate genes and anonymous and genetically an-
chored markers have been constructed. (Foote et al.,
1992; Chumakov et al., 1992; Collins et al., 1995; Bell
et al., 1995). However, the YAC clones that have been
used to create these maps are often unstable and chi-
meric in nature (Green et al., 1991; Kouprina et al.,
1994; Larinov et al., 1994). Further, it is difficult to
recover the DNA cloned into YACs in a pure form.
Therefore it is necessary to develop alternative re-
sources that retain the advantages of large insert size
and yet are stable, facile to manipulate, and apply stan-
dard techniques in molecular biology.
The BAC cloning system is capable of stably propa-
1
To whom correspondence should be addressed.
213
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gating complex DNA inserts in Escherichia coli (Shi-
zuya et al., 1992). The basis for the high degree of sta-
bility of DNA fragments containing repetitive se-
quences is the single-copy number of the vector (Kim
et al., 1992; Shizuya et al., 1992; Iaonnou et al., 1994).
BAC libraries with inserts averaging around 130–150
kb are well suited to bridge the gap between higher
order genome maps, including those based on overlap-
ping YACs or STS order, and the need to perform de-
tailed analysis of specific clones. Thus, BAC libraries
serve to integrate genetic, STS, and cytogenetic map
information while providing direct access to stable ma-
terial that may be utilized for the purpose of molecular
analyses, medical diagnostics, and ultimately for geno-
mic sequencing.
We have previously constructed human BAC clones
with inserts larger than 300 kb and demonstrated the
stable propagation of human DNA using this cloning
system (Shizuya et al., 1992). BAC libraries con-
structed from plants (Wang et al., 1995; Woo et al.,
1994) as well as from an individual human chromosome
(Wang et al., 1994) have been reported recently. We
have developed a new BAC cloning vector that permits
efficient BAC cloning. This report describes the vector
and the characteristics and utility of the human BAC
library.
MATERIALS AND METHODS
Library construction. The pBAC108L vector was described pre-
viously. pBeloBAC11, which allows identification of transformants
with and without inserts by colony color through a complementation
of the lacZ gene, was constructed by ligating a BstUI minor fragment
from pGEM3z (Promega) containing the lacZ region with BamHI and
HindIII cloning sites to the NotI major fragment from pBAC108L
(Shizuya et al., 1992) via NotI linkers. Vector DNA was purified by
CsCl–EtBr differential centrifugation, cut with HindIII, dephos-
phorylated by HK-phosphatase (Epicentre), and used for ligation.
High-molecular-weight DNA was prepared in agarose from the
human male fibroblasts CCD-978SK (ATCC No. CRL 1905). The cells
were harvested at low passage number and embedded in agarose,
and the DNA was purified as described previously (Birren and Lai,
1993). Detailed methods for BAC library construction are described
elsewhere (WEB page http://www.tree.caltech.edu; Birren, et al., in
press). Briefly, the embedded DNA was partially digested with Hin-
dIII and size selected by two rounds of pulsed-field electrophoresis
0888-7543/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
gnma AP: Genomics
214 KIM ET AL.

with a 90-s switch time at a 1207 angle to minimize the content of

small molecules. After digestion of the agarose with Gelase (Epicen-
tre), the size-selected DNA was ligated to the HindIII-digested and
dephosphorylated vector at an approximately 10:1 molar ratio of
insert DNA:vector. A typical ligation reaction of 120 ml contained
100 ng of insert DNA (added as 100 ml at 1 ng/ml) of 200-kb average
size, 36.5 ng of vector, and 200 units of T4 DNA ligase (New England
Biolabs). After incubation overnight at 167C, ligations were dialyzed
for 2 h at room temperature against 0.51 TE and electroporated into
the host cells DH10B (using 3 ml of ligation / 25 ml of cells) as
described (Sheng et al., 1995). Cells were allowed to recover by shak-
ing for 45 min at 377C in SOC medium and were plated on LB agar
plates containing 12.5 mg/ml chloramphenicol and, when pBelo-
BAC11 was used, 50 mg/ml X-Gal and 25 mg/ml IPTG. After 24 h of
growth at 377C, white and blue colonies were clearly distinguishable.
The construction of the entire library entailed the preparation of 5
separate partial digestions of DNA and approximately 70 ligations
and transformations. Quality controls of the batches of vector and
competent cells used were routinely performed. The number and
average size of the clones obtained from each transformation are
reflections of many factors, among which concentration, size, and
integrity of the insert DNA that was used were the most critical and
difficult to control. Therefore before selecting the clones for inclusion
in the library, a sampling of clones from each transformation was
analyzed to ensure that an acceptable proportion of clones contained
inserts and that the inserts were of a suitable average size.
Library screening. The library arrayed in 1000 96-well microtiter
plates was prepared to allow efficient screening by both colony hy- FIG. 1. Diagram of pBeloBAC11 vector. The lacZ gene fragment
bridization and PCR. High-density colony grid filters were prepared was introduced to the NotI site of pBAC108L. Insertional inactiva-
as described previously (Kim et al., 1994). To increase the efficiency tion of the gene by a cloned DNA fragment results in white colonies
of library screening by hybridization, multiple probes were combined on X-gal/IPTG plates after transformation.
(usually 10–50) and used for each hybridization against the entire
library. Library pooling for PCR screening was performed as illus-
trated in Fig. 4A. First, groups of 20 plates from the library were bridization using labeled human genomic DNA as
pooled, producing 50 different large pools. For each of these large probe. The remainder of the library was constructed
groups of 20 plates, smaller pools representing 2 consecutive microti- using the vector pBeloBAC11 (Fig. 1). This new vec-
ter plates (one odd-numbered plate and one even-numbered plate), tor uses a complementation of the lacZ gene to indi-
column pools (A1 to H1 positions, etc.) from all 20 plates, and row
pools (A1 to A12 positions, etc.) from the stacks of 10 odd-numbered
cate recombinant molecules through colony color. As
plates and 10 even-numbered plates were generated. To screen the with the pBAC108L vector, the inserts can be excised
library for an STS, first 50 large pool samples were PCR amplified, from the vector by NotI digestion. The clones can be
and the products were assayed on a gel for the presence of the correct linearized in vitro by using l terminase that recog-
DNA band. The large pools exhibiting correct PCR products were nizes and introduces a staggered cut specifically at
further analyzed by PCR amplification of the corresponding 38 sub-
pools. As shown in Fig. 4B, PCR products from 2-plate pools, column the cosN site, generating two cohesive ends that are
pools, and odd and even row pools were assayed on agarose gels, and complementary to each other but nonidentical (Feiss
the addresses of the clones containing the STS were deduced. et al., 1983). These cohesive ends can be specifically
Miniprep of BAC DNA. Clones were grown in 3 ml LB containing labeled using complementary oligonucleotides and
chloramphenicol (12.5 mg/ml) and miniprepped either manually ac- used for restriction mapping of BAC inserts (not
cording to the alkaline lysis method (Sambrook et al., 1989) or by shown). The loxP site also permits linearization as
using an automated miniprep machine (AutoGen740, Integrated
Separation Systems). Each lane in Fig. 5 contains DNA representing well as introduction of additional DNA segments via
0.75 ml culture. Cre-mediated recombination (Heiss et al., 1982).
All the human BAC clones described in this paper are available The library was constructed in a series of approxi-
upon request. Recently, an additional 160,000 human BAC clones mately 70 separate transformations, often involving
or 61 libraries have been produced and are commercially available
different batches of ligation products that resulted in
through Research Genetics, Inc. (Huntsville, AL).
varying degrees of efficiency and quality of the clones
generated (see Materials and Methods). Overall, based
RESULTS AND DISCUSSION on quality checks performed during library construc-
tion as well as our library screening, we estimate that
We have constructed a human BAC library con- at least 85% of the clones in the library contain inserts.
sisting of 96,000 clones arrayed in 1000 96-well Figure 2 illustrates the size distribution of BAC clones
plates. The clones were produced using HindIII par- containing inserts as determined by NotI digestion and
tially digested DNA from primary fibroblasts from a pulsed-field gel electrophoresis. These clones include a
normal male. The initial portion of the library, 399 random sampling of clones that have been identified
plates, was constructed using pBAC108L vector during the library screens. We estimate the average
(Shizuya et al., 1992). Clones in this vector con- insert size of BAC clones in the library that contain
taining human inserts were identified by colony hy- inserts to be 140 kb. Estimating from the insert size

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 CONSTRUCTION AND CHARACTERIZATION OF A BAC LIBRARY
FIG. 2. Clone insert size distribution. The 406 BACs with known
insert size were plotted against the size ranges. Clones without in-
serts (approximately 15% of the total) are not included.
and the percentage of clones with inserts, we predict
the coverage of the library to be approximately 41 of
human genome. This is in good agreement with results
obtained from screening the library with unique hu-
man probes such as cDNA and STS markers (Table
1), as well as with entire cosmids under suppressive
hybridization conditions. More than 92% of the probes
that have been used to screen the library identified one
or more hits. This is close to the 98% frequency pre-
dicted by a Poisson distribution for recovery of any
marker from a 41 library. With this relatively small
number of markers used, as well as the unknown rate
of false negative results associated with each of these
screening methods, it is not clear whether the apparent
discrepancy between these two values, 92 vs 98%, could
reflect a small degree of nonrandom representation in
the library. However, with 375 different probes, on av-
erage 3.9 positive clones per marker were identified in
our screens (Table 1).
Highly efficient screening methods are required to
use arrayed libraries of this size for construction of
contigs. BACs are amenable to screening by either of
two independent methods, hybridization or PCR. To
TABLE 1
Summary of BAC Library Screening with Unique Genomic Markers
Type of Method of No. of markers
markers screening screened
cDNA Hybridization 93
STS PCR 105
Cosmid Hybridization 177
Note. For each type of marker, the total number of markers used, the method of library screening, the total number of positive BACs
identified, the average number of hits per marker, and the number and percentage (shown in parentheses) of the markers that did not find
any positive clones from the library are shown.
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215
allow efficient hybridization screening, the BAC library
was gridded at high density onto nylon filters (Kim et
al., 1994). One advantage of hybridization is the ability
to combine probes to screen the entire library simulta-
neously with multiple probes. In this way, all BACs
derived from an entire region can be identified in a
single experiment. Figure 3 shows an example of the
BAC library screened with a mixture of 20 cosmid
clones. To determine which specific hybridization sig-
nals resulted from particular individual probes in the
mixture, a subsequent round of hybridization is per-
formed. This can be carried out by preparing a number
of new filters, each containing the positive clones from
the initial screen, and hybridizing with the individual
probes (Fig. 3B). If a portion of each labeled probe is
reserved from the individual hybridization (i.e., not
mixed with other probes), then only a single round of
labeling is required to complete the screens. High-den-
sity BAC colony filters could be screened by a variety
of probes, including unique DNA markers (Kim et al.,
1994, 1995a), cosmids (Kim et al., 1994) (Fig. 3), P1
clones (not shown), BAC inserts, Alu-PCR products
(unpublished), YACs and amplified products from
YACs (unpublished), and an entire flow-sorted chromo-
some (Kim et al., 1995a).
The BAC library was also pooled to permit PCR
screening for STS content according to the scheme
shown in Fig. 4A. The strategy involves two levels
of screening; for the first, 50 large pools were gener-
ated by combining all the clones from concentrated
sets of 20 96-well plates. Thus each of these pools
contains 1920 clones or one-tenth of a genome equiv-
alent, a level of complexity far below the limit for
PCR screening of BACs (unpublished). From each
large pool, a set of 38 subpools was created, with 10
of these representing the contents of 2 plates (the
odd/even pools), as well as 16 row pools (8 row pools
from odd-numbered plates and 8 row pools from
even-numbered plates) and 12 column pools. After
growth of the individual clones in 96-well plates,
BAC DNA was prepared from pooled bacterial cul-
tures using the Autogen 740 miniprep machine.
Identification of clones in the library containing a
marker using this scheme requires first screening
the 50 large pools and then performing an additional
Total No. Average No. No. of markers
of hits of hits without hit (%)
380 4.1 7 (7.5%)
378 3.6 9 (8.6%)
706 4.0 13 (7.3%)
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216 KIM ET AL.

FIG. 3. Library screening by hybridization. (A) Twenty cosmid probes were pooled and used for screening the entire library. Only 41 of
the library is shown. Signals from more than 50 positive colonies are evident. (B) Screening the positive clones from (A) with individual
probes. BACs positive for single cosmid probes were identified on the filters containing a group of BACs that are positive to pooled probes.
The autoradiogram shows 6 strong signals that indicate positive clones.

38 PCR analyses for each positive large pool. Typical
results from this screening are shown in Fig. 4B.
We have previously demonstrated the stability of
large human DNA inserts in single-copy bacterial clon-
ing systems that have been developed in our laboratory
(Kim et al., 1992; Shizuya et al., 1992). The stability
of the clone inserts not only offers reliability for the
materials for genome analysis and DNA sequencing
but also contributes to fuller representation of the ge-
nome in the library. A gap in the P1 contig from the

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 CONSTRUCTION AND CHARACTERIZATION OF A BAC LIBRARY 217

FIG. 4. (A) Library pooling scheme. For details, see Materials and Methods. (B) PCR screening of the library for STS326. PCR products
were separated on 2% agarose gel and stained with ethidium bromide. In this example, there are five BACs containing STS326 since five
of the large pools show PCR-amplified DNA bands of the expected size. One of the positive large pools was chosen to demonstrate how the
address of the positive clone is deduced from the subpool PCR. Note the PCR bands from a two-plate pool, a row pool, and a column pool.
M and C indicate molecular weight markers and control PCR products from human placental DNA, respectively.

human BRCA1 locus could be filled by BAC clones library screens are derived from the same region, it can
(Neuhausen et al., 1994). Because BACs are several indicate the extent of overlap between the clones, and
times larger than cosmids, they provide relatively effi- it can indicate the order of clones within the contig. In
cient means to construct physical contig maps. Finger- addition, comparing restriction fragments from inde-
print analysis data for chromosome 22-specific BAC pendent clones enables detection of rearrangements or
clones indicate that BACs are nearly free of chimerism deletions involving segments as small as a few kilo-
(not shown). In addition, of the 82 BAC clones that were bases. The fact that we do not observe such rearrange-
analyzed by FISH mapping, 8 clones showed positive
signals at more than one chromosomal locus (Kim et
al., 1995b). While it is possible that these clones are
true chimeras, some of the multiple signals could be
attributed to the reiterative nature of the human ge-
nome. Multiple signals are also caused by occasional
clone mixes during library arraying and replication
(not shown).
BAC DNA that can be readily prepared by both man-
ual and automated procedures can be directly used for
applications such as FISH mapping, restriction fin-
gerprint analysis, and generation of small-insert subli-
braries for random shotgun sequencing the BAC clones.
BAC clones can be analyzed in greater detail using
DNA purified by alkaline lysis methods. Bacterial cul-
ture volumes of less than 1 ml yield sufficient DNA for
analysis of BAC restriction fragments using agarose
gel electrophoresis and ethidium staining. Figure 5
shows HindIII digestion patterns of a group of overlap- FIG. 5. HindIII digests of a group of overlapping BACs. Fifteen
ping BAC clones obtained from this library that cover BAC clones were selected from a BAC contig on chromosome 22.
Approximately a quarter of the miniprep DNA obtained from 3-ml
a region spanning approximately 1000 kb. This restric- cultures was digested with HindIII and resolved on 0.7% agarose gel.
tion analysis of BAC clones serves several functions; Bacteriophage l DNA digested with HindIII was used as a molecular
it allows rapid confirmation that clones identified in weight marker (M).

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218 KIM ET AL.
ments in overlapping BACs suggests that the se-
quences are being maintained with a high degree of
fidelity. Further, this analysis will reveal the presence
of chimeric clones through the presence of distinct sets
of restriction fragments derived from unrelated geno-
mic regions. Our restriction analysis of overlapping
clones from this library indicates a low frequency of
chimerism among these clones (on the order of 5%, un-
published).
Their large insert size, high stability, ease of manipu-
lation, and high genomic coverage suggest that BACs
are ideal building blocks for high-resolution physical
maps over the entire genome. Currently cosmids are
the most frequently used substrates for genomic se-
quencing. Given that the average size of a BAC clone
is equivalent to that of typical cosmid contigs that con-
sist of 5–10 cosmid clones, it is reasonable to hypothe-
size that BACs will provide vastly more efficient sub-
strates for major genomic sequencing efforts. Recently,
by sequencing five BAC clones obtained from this li-
brary, it was possible to determine the complete se-
quence of approximately 650 kb from the human T cell
a/d locus (C. Boysen, unpublished). Taken together, the
BAC library that we have described here will serve as
an invaluable tool for human genomic studies at all
levels.
ACKNOWLEDGMENTS
We thank April Mengos, Ana Mercante, and Yu-Ling Sheng for
their excellent technical assistance. This work was supported by De-
partment of Energy Grant FG0389ER60891 funded to M.I.S.
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