441

Genomic DNA Libraries,

Construction and Applications

Eugene R. Zabarovsky
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden

1 Principles 443

2 Techniques 445
2.1 General Characteristics of λ-based Vectors Used for Construction of
Genomic Libraries 445
2.2 Construction of General Genomic Libraries 447
2.3 Construction of Jumping and Linking Libraries. Use of Linking
and Jumping Clones to Construct a Physical Chromosome Map 451

3 Applications and Perspectives 455

3.1 Cloning DNA Markers Specific for a Particular Chromosome 455
3.2 CpG Islands as Powerful Markers for Genome Mapping;
CpG Islands and Functional Genes 457
3.3 Alu-PCR and Subtractive Procedures to Clone CpG Islands
from Defined Regions of Chromosomes 458
3.4 IBD (Identical-by-descent) Fragments for Identification
of Disease Genes 460
3.5 Strategies to Map and Sequence Genomes; Hierarchical, Whole-genome,
and Slalom Sequencing Approaches 460
3.6 Restriction-site-tagged Microarrays to Study CpG-Island Methylation 462
3.7 Restriction-site-tagged Sequences to Study Biodiversity 464

4 Summary 465

Bibliography 465
Books and Reviews 465
Primary Literature 465

Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Edited by Robert A. Meyers.
Copyright  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
ISBN: 3-527-30547-5
442 Genomic DNA Libraries, Construction and Applications

Keywords
Blue–white Selection
Not really selection but color identification. Vectors carrying the β-galactosidase (lacZ)
gene (or part of it) produce blue plaques in the presence of 5-bromo-4-chloro-3-indolyl-
β-D-galactopyranoside (X-gal). If this gene is located in a stuffer fragment, then all
recombinants will form white plaques and parental vectors will produce blue plaques
in the presence of X-gal.

Genetic Selection
Usually, in cloning, selection against parental, nonrecombinant molecules in favor of
the recombinant. For λ-based vectors used for construction of genomic libraries, the
two most commonly used types of selection are Spi and supF. Spi+ phages carrying red
and gam genes cannot grow in E. coli lysogens carrying prophage P2; since, however,
the majority of the vectors contain these genes in a stuffer fragment, only recombinant
phages can grow in such E. coli strains. Selection for supF exploits λ vectors carrying
amber mutations. These vectors cannot replicate without the supF gene, which must be
present either in the host or in the cloned insert. If the insert carries the supF gene,
only recombinant phages will be able to replicate in an E. coli host without the
suppressor gene.

Polylinker
A short DNA fragment (in the vector) that contains recognition sites for many
restriction enzymes, which can be used for cloning DNA fragments into this vector.

Restriction Enzyme
An enzyme that recognizes a specific sequence in DNA and can cut at or near this
sequence. In cloning procedures, the most commonly used enzymes produce specific
protruding (sticky) ends at the ends of the DNA molecule. Each enzyme produces
unique sticky ends. The DNA molecules possessing the same sticky ends can be
efficiently joined with the aid of DNA ligase (see ligation).

(STS) Sequence-tagged Site

A short (200–500 bp) sequenced fragment of genomic DNA that can be specifically
amplified using PCR. STS represents or is linked to some kind of marker (i.e. it is
mapped to a specific locus on a chromosome).

By virtue of the powerful technology developed in molecular biology, it is pos-

sible to isolate any DNA fragment in the genome of an organism and, after
reverse transcription, any transcribed gene in the form of a complementary DNA.
The isolation (cloning) procedure involves the insertion of the DNA fragment
into a vector, capable of replication in a microorganism, which allows production
of large quantities of the DNA fragment for physical or biological analysis. Upon
determination of the location in the genome from which the particular DNA
 Genomic DNA Libraries, Construction and Applications 443

fragment was derived, that fragment acquires the property of a DNA marker. Such
DNA markers are a prerequisite for physical and genetic mapping of the genome
of the organism. DNA markers are also of importance for the diagnosis of genetic
diseases. DNA markers can be divided into several different classes depending on
the way in which the markers were selected among the fragments of genomic DNA.
Examples of such classes are anonymous, micro- and minisatellites, restriction
fragment length polymorphism (RFLP) markers, and NotI linking clones.
Vectors and clone libraries of different types can be used to clone markers.
Lambda-based vectors and genomic libraries of different kinds are commonly used
for this purpose. Many different variants of λ-based vectors that combine features
of different cloning vehicles (plasmids, M13 and P1 phages) have been created
for this purpose. The use of each vector is usually limited to a specific task:
the construction of general genomic libraries (which contain all genomic DNA
fragments) or special genomic libraries (which contain only a particular subset of
genomic DNA fragments). Among these special libraries, NotI linking and jumping
libraries have particular value for physical/genetic mapping and sequencing of the
human genome. Shotgun and slalom libraries are usually used for sequencing
purpose and comparative genomics.

1 concentrates on the widely used λ-based

Principles
In molecular biology, ‘‘cloning’’ is the
insertion of DNA with interesting infor-
mation into a specific vector that allows
replication and transfer of the cloned DNA
from one host to another. The vector
containing the inserted DNA is called a
recombinant vector to distinguish it from
its parental vector, which does not contain
any foreign DNA. Usually, ‘‘interesting in-
formation’’ is a piece of DNA obtained
from any target organism; it can be a gene
(or part of a gene) or simply anonymous
DNA sequences for which no function is
yet known. It can originate directly from
DNA or can be obtained from reverse tran-
scription of RNA molecules. The main
idea of cloning is to obtain the interesting
piece of DNA in a quantity large enough
for analysis and further experiments. Now,
the vectors and strategies used for cloning
come in many different types. This chapter
vectors and the construction of genomic
libraries, which played an important role
in the Human Genome Project.
A genomic library is a collection of
recombinant vectors; it contains DNA
fragments representing the genome of
a particular organism. Genomic libraries
can be either general, containing DNA
fragments covering the whole genome,
or special, containing only specific ge-
nomic fragments that differ in certain
parameters. Some are CG rich whereas
others contain only particular size frag-
ments of DNA obtained after digestion
with a particular restriction enzyme, con-
tain specific repeats, and so on. Important
special genomic libraries are the jumping
and linking types (see Sect. 2.3).
Cloned DNA fragments can be located
to a specific site of a chromosome, af-
ter which they can serve as markers for
physical and genetic mapping. Different
types of markers are used. The so-called
444 Genomic DNA Libraries, Construction and Applications

anonymous markers represent randomly point mutation in some individuals of a

cloned DNA fragments whose functions
or specific features are not known. Other
DNA markers can possess specific fea-
tures. They can contain a known gene or
expressed sequences with unknown func-
tion, CpG islands (also associated with
genes, see Sect. 3.2), or recognition sites
for rare cutting restriction enzymes con-
venient for long-range mapping. Such
markers can be polymorphic, that is, they
have different structures in different indi-
viduals (they are usually distinguished on
the basis of different mobility in gel elec-
trophoresis). Such markers are extremely
important in mapping and cloning human
disease genes and for construction of ge-
netic maps. Three types of polymorphic
markers are commonly used (Fig. 1).
Single-nucleotide polymorphism (SNP)
markers are DNA fragments that have a
population. The advantages of SNPs are
their abundant numbers (>106 ) and the
fact that they can be detected by nonelec-
trophoretic methods, for example, using
microarrays. However, usually SNP has
only two alleles. A subtype of SNPs are
RFLP markers that recognize genomic
fragments containing polymorphic recog-
nition sites for a particular restriction
endonuclease (e.g. TaqI, MspI). The same
chromosomal regions in different individ-
uals contain or lack this recognition site.
A second form of DNA polymorphism
results from variation in the number
of tandemly repeated (VNTR) DNA se-
quences in a particular locus. Usually,
they are divided in two types – mini- and
microsatellites. Minisatellites are DNA
fragments 0.1 to 20 kb long that contain
many copies (from 3 to more than 40) of

Bam HI Bam HI Bam HI

RFLP 2 kb 4 kb
markers
Allele 1

Bam HI Bam HI Bam HI

Allele 2

6 kb
VNTP
markers Repeats

Allele 1

Bam HI 0.6 kb Bam HI

Repeats
Allele 2

Bam HI 1.4 kb Bam HI

Repeats
Allele 3

Bam HI 1.6 kb Bam HI

Fig. 1 The difference between RFLP and VNTR polymorphism.
 Genomic DNA Libraries, Construction and Applications
6 to 60 bp repeats. All these repeats share
a 10 to 15 bp core sequence similar to the
generalized recombination signal (chi) of
E. coli. When DNA from different individ-
uals is digested with a restriction enzyme
that does not cut inside these repeats, the
length of the fragments produced will de-
pend on the number of repeats at the
locus. Since the minisatellites and re-
peats constitute a relatively large fragment,
it is possible to discriminate between
different alleles using ordinary nondena-
turing gel electrophoresis and Southern
blot analysis. Many different alleles (usu-
ally more than five) can be distinguished
at a locus containing minisatellites. Min-
isatellites cluster around the distal ends
of human chromosomes and, sometimes,
are located near the genes.
Microsatellites are relatively short DNA
fragments (usually <100 bp) with repeat
units from 1 to 5 bp (such as A, AC,
AG, AAC, AAAG, etc.). They are numer-
ous (5 × 105 ) and uniformly distributed
throughout the human genome with an
estimated average spacing of about 6 kb.
Microsatellites can be very polymorphic
(more than 10 alleles at the same locus),
and polymorphism usually increases with
the number of repeats. Microsatellites with
fewer than 10 copies are usually not poly-
morphic. Since microsatellites are short,
they can be analyzed quickly by using
the polymerase chain reaction (PCR) with
primers flanking each locus. Different al-
leles can be resolved using denaturing
gel electrophoresis. Very frequently, mi-
crosatellites are associated with Alu repeats
(see Sect. 3.1) and this creates problems for
their use with PCR, since Alu repeats are
conserved in the human genome. Thus,
flanking primers for the microsatellite
located in the Alu repeat are unlikely to be
useful because they prime from many Alu
repeats and, instead of discrete bands, give
445
a smear after electrophoretic separation of
the PCR products.
An optimal marker should have the fea-
tures of all the different types of markers
just discussed. The ideal marker should
contain (1) a gene, (2) a CpG island that
has been shown to be very conserved in
the genome and can be used for compar-
ative gene mapping in different species,
(3) a rare cutting restriction site useful for
physical mapping, and (4) polymorphic se-
quences (e.g. microsatellites). One of the
best candidates for having all these fea-
tures together is NotI linking clones, that
is, recombinant clones containing the NotI
restriction site.
2
Techniques
2.1
General Characteristics of λ-based Vectors
Used for Construction of Genomic Libraries
Among other more modern vectors used
for construction of genomic libraries
(yeast, bacterial and P1 artificial chromo-
somes; YAC, BAC, and PAC respectively),
phage λ-based vectors are still very pop-
ular. The reason is that the genetics and
features of both λ phage and E. coli (the
host for λ phage) are well known. The
size of the phage DNA that can be pack-
aged into viable phage particles is limited
between 37.7 and 52.9 kb. This means
that it is possible to biologically regulate
the size range of the cloned DNA frag-
ment. There exist extremely efficient in
vitro systems for packaging such DNA
into λ phage particles (109 plaque-forming
units per µg of DNA) to produce vi-
able phages. To combine the features of
different vector systems, extensive modifi-
cations of λ phage vectors were developed
(Fig. 2).
 446
T7 SP6

XbaI
Eco RI
Avr II
BamHI

Sfi l
XhoI
BamHI
EcoRI
XbaI
XhoI
SacI
Sfi I

Avr ll
XbaI
SacI
XhoI
Bam HI
Hind III
Sfi I
Nae I
Not I
Xho I
Eco RI
Avr ll
XbaI
XbaI
Avr II
EcoRI
XhoI
Not I
NaeI
Sfi I
Hind III
Bam HI
XhoI
Sac I
XbaI

Sac I
λ GEM-11 14.0 9.2 λSK6 9.2
13.0
20.3 Sal I 20.3 lacZO SalI lacZO
T3 T7

Sac I
KpnI
Sm aI
NcoI
Nhe I
Avr II
Sfi I
NaeI
Not I
XhoI
EcoRI
BamHI
Sal I
Sal I
BamHI
EcoRI
XhoI
Not I
NaeI
Sfi I
Avr II
NheI
NcoI
XbaI

XbaI
XhoI
Sac I
Hind III
BamHI
Eco RI
Not I
Sal I
XbaI

XbaI
Sal I
Not I
EcoRI
BamHI
Hind III
Sac I
XhoI
XbaI
Mst II
ClaI BspMlI
λ DASHII 13.0 9.2 λSK17 13.0 XhoI

Sal I lacZ 12.7

20.3 Sal I 20.3 Sal I ClaI SalI Bs pMlI
T3 ApR
T7
pBR322 M13
Genomic DNA Libraries, Construction and Applications

ori 3.9 ori

XbaI
Eco RI
Xho I
Sal I
Sac I
Not I
Sac I
XbaI

XbaI
Sac I
Not I
Sac II
Sal I
Xho I
Eco RI
XbaI
XbaI
BamHI
Xho I
Eco RI
Avr II
Nae I
Nae I
Avr II
Eco RI
Xho I
BamHI
SmaI
KpnI
SacI

λ FIXII 9.2 BspMlI

13.0 ClaI BspMlI ClaI
λ SK40 13.0
20.3 Sal I
20.3 Mst II 9.2
Sal I ApR

Sal I
Bam HI
Eco RI
Eco RI
Bam HI
Sal I
pBR322 M13
ori 3.9 ori
Sal I
I-SceI
SpeI
Not I
XmaIII
XmaIII
Not I
SpeI
I-Scel
SalI

λ EMBL3 13.0 9.2

20.3 Sal I
Fig. 2 Examples of λ-based vectors: standard λ vectors (λGEM11, λFIXII, λDASH, λEMBL3, λSK6) and diphasmid vectors (λSK17 and λSK40). Sizes
are in kilobases. Not all restriction sites are shown. Heavy lines represent vector arms, thin lines denote the stuffer fragment; open boxes mark plasmid
and M13 sequences; lacZO is the lac operator sequence. T3, T7, SP6 – promoters for T3, T7, and SP6 RNA polymerases.
 Genomic DNA Libraries, Construction and Applications
Cosmids are essentially plasmids that
contain the cos region of phage λ
responsible for packaging of DNA into
the phage particle. The advantages of cos-
mids are easy handling (as with plasmids)
and large cloning capacity. Since the plas-
mid body is usually small (3–6 kb), large
DNA molecules (46–49 kb) can be cloned
in these vectors.
Phasmids are λ phages that have an
inserted plasmid. They have the same ba-
sic features as λ phage vectors, but the
inserted foreign DNA fragment can be
separated from the body of the phage DNA
and converted into the plasmid form. After
the conversion, the cloned DNA fragment
will exist as a recombinant plasmid.
Hyphages represent another type of λ-
based vectors. They were constructed from
M13 vectors with a built-in cos site of λ.
Since these vectors have the main features
of M13 vectors, they can be obtained in
single-stranded form. Their distinctive fea-
ture is the capability to be packaged with
high efficiency into λ-phage-like particles.
This decreases the chance of recovering
nonrecombinant vectors and opens the
possibility of constructing a representative
genomic library in single-stranded form.
Diphasmids are vectors, which offer the
opportunity to combine the advantages of
phages (λ and M13) and plasmids. Diphas-
mids can be divided into two classes: those
that can replicate as phage λ (an improve-
ment over phasmids) and those that are
incapable of replicating as phage λ (i.e.
a cosmid capable of being packaged into
phage M13 particles).
In some cases, it is more convenient to
work with a genomic library in plasmid
than in λ phage form. The construction of
a representative genomic library directly
in a plasmid vector has several drawbacks
and difficulties. However, all these prob-
lems can be easily solved with the help
447
of phasmid and diphasmid vectors. Often
a genomic library is constructed in a λ
phage and then converted in its entirety to
plasmid form.
2.2
Construction of General Genomic Libraries
Representativity is one of the most im-
portant features of a genomic library. In a
‘‘representative’’ genomic library, every ge-
nomic DNA fragment will be present in at
least one of the recombinant phages of the
library. In practice, however, this is diffi-
cult to achieve. Some genomic fragments
are not clonable because of the strategy
used for construction of the genomic li-
brary. For example, if the maximal cloning
capacity of the vector is 18 kb and EcoRI
digestion is used to construct the library,
no genomic EcoRI fragments bigger than
18 kb will be present. In some cases, ge-
nomic DNA fragments can suppress the
growth of the vector or the host cell, with
the result that its cloning can be restricted
to specific vector systems.
The important reason for decreased
representativity is the different replication
potential of different recombinant phages.
Most researchers work with amplified
libraries. The ligated DNA molecules
are packaged into the λ phage particles
and plated on a lawn of E. coli cells
(usually many petri dishes are used for
such plating). Then all λ phage particles
are eluted from the plaques, and the
liquid eluted from all the petri dishes is
mixed. Glycerol or dimethyl sulfoxide is
added to the eluate, and the aliquots are
kept at – 76 ◦ C. This procedure is called
amplification of the library.
With amplification, a library can be kept
for many years and can be used for many
experiments by many researchers. On
the other hand, each recombinant phage
present in the library gives a single plaque
448 Genomic DNA Libraries, Construction and Applications
at the first plating, and since different
recombinant phages differ in their growth
potential, there may be differences of 100
times in the abundance of clones after
amplification. This means that in the
amplified library, some of the phages are
present 100 times more often than others.
In this case, to recover all recombinant
phages obtained after packaging into λ
phage particles, one needs to plate 100
times more phages than that obtained after
the original plating. Since such quantities
are difficult to achieve in practice, some
recombinant phages are virtually lost from
the library after amplification.
How is the representativity of a library
estimated? A library is considered to be
representative if after the first plating (be-
fore amplification) it contains a number
of recombinant clones together, contain-
ing genomic DNA fragments equal to 7
to 10 genome equivalents. For example,
human genomic DNA contains approx-
imately 3 × 109 bp. If the vector contains
on an average 15 kb inserts, the representa-
tive library should contain 1.4 to 2.0 × 106
recombinant clones.
The way in which the genomic DNA
fragments are produced for cloning is
also important. The more randomly the
genomic DNA is broken, the more repre-
sentative a library can be obtained. Clearly,
the EcoRI enzyme (6 bp recognition site)
will cut genomic DNA less randomly than
Sau3AI (4 bp recognition). Probably, the
shearing of DNA molecules using phys-
ical methods (e.g. syringe, sonication) is
the most reliable way to obtain randomly
broken DNA molecules.
An important characteristic feature of a
library is the percentage of recombinants.
For most purposes, if a library contains
more than 80% of recombinant phages,
it is better to omit the genetic selection
procedure because it usually decreases the
representativity of the library. To calculate
the percentage of recombinants, one can
use genetic (Spi) selection as in the case of
λEMBL vectors. Another approach relies
on blue–white color identification (e.g. λ-
Charon series). A third class of vectors
has both genetic selection and blue–white
color identification (λSK4, λSK6). There
are three commonly used ways to construct
genomic libraries (Table 1, Fig. 3).
The original (‘‘classical’’) method in-
cludes generation of sheared genomic
DNA fragments using physical or en-
zymatic manipulations followed by the
physical separation of fragments of a
particular size using, for example, ultra-
centrifugation or gel electrophoresis. The
vector DNA is digested with two (or even
three) restriction enzymes whose recogni-
tion sites are located in the polylinker and
are separated by a few base pairs. The arms
and the stuffer piece are purified from the
small oligonucleotide molecules released
after the digestion by, for example, pre-
cipitation with polyethylene glycol (PEG)
6000. The stuffer piece and both arms will
now have different sticky ends, preventing
re-creation of the original vector molecules
during subsequent ligation with genomic
DNA fragments.
In the second ‘‘dephosphorylation’’ ap-
proach, the phage arms are prepared by si-
multaneous digestion with two restriction
enzymes as shown earlier. Genomic DNA
is partially digested to the extent that DNA
fragments with sizes in the range of 15
to 20 kb will represent the majority of the
products. These DNA fragments are de-
phosphorylated to prevent their ligation to
each other and are then ligated to the vector
arms. If too big or too small genomic DNA
fragments are ligated to the phage arms,
size limitations will make it impossible for
these recombinant molecules to yield vi-
able phages. Compared to the preceding
Tab. 1 Comparison of three basic methods used in construction of representative genomic libraries.

Method DNA [µg] DNA Self-ligation of Effectiveness of packaging Number of Genetic selection
needed to fractionation packaging necessary to remove
construct a vector genomic Per Per reactions to get nonrecombinants
representative DNA DNA microgram of microgram of representative
genomic vector DNA genomic DNA library at
library maximal
efficiency
per microgram
of genomic DNA

Classical 100–1000 Yes Yes Yes 105 –107 105 –107 1 Yes
Dephosphorylation 5–10 No Yes No 104 –105 105 –106 3–5 Yes
Partial filling in 5–10 No No No 105 –107 105 –107 1 No
Genomic DNA Libraries, Construction and Applications
449
450 Genomic DNA Libraries, Construction and Applications

(a) λEMBL 3 Eukaryotic genomic DNA

SB R RBS
cos L R cos
Left arm Right arm Partial digestion with
Digestion with Sau 3AI, isolation of
Bam HI + Eco RI DNA fragments
cos L B B R cos 15–20 kb in length
R R
Stuffer
Ligation with
T4 DNA ligase
R cos L R cos L R cos L

Packaging in vitro
into λ phage
particles

λ SK5 Eukaryotic genomic DNA

(b) BXSR RSXB
cos L R cos
Left arm Right arm
Digestion with Partial digestion
Bam HI + EcoRI with Sau 3AI
cos L S S R cos
R R
Stuffer
piece Partial filling-in of
dCTP termini with Klenow dATP
dTTP fragment of E. coli dGTP
DNA polymerase
cos L GTC-3´
CAGCT-5´ Ligation with 5´-GATC GA-3´
3´-AG CTAG-5´
T4 DNA ligase
5´-TCGAC R cos
3´-CTG
R cos L R cos L R cos L

Packaging in vitro
into λ phage
particles

Fig. 3 Two approaches for constructing genomic libraries:

(a) classical method and (b) partial filling-in method. Lcos and
Rcos – left and right parts of the cos site correspondingly; B, BamHI;
R, EcoRI; S, SalI; X, XmaIII.
 Genomic DNA Libraries, Construction and Applications 451

ori
M13
ori Ampr R
lacI S
SK18 B
A
lacZ H
R L

A. Digest with Hind III cos B. Digest with Eco RI

SBAH
RSBA
Ampr Ampr
R L R L
5´-AGCTT A-3´ lacI G-3´
5´-AATTC
3´-A cos ori TTCGA-5´ 3´-G cos GTTAA-5´
lacZ ori lacI ori ori
Partial filling-in lacZ
M13 M13
dATP

Digest with Bam HI AH

RS
Ampr Ampr
R L R L lacI
5´-AGCTT 5´-GATCC GAA-3´
G-3´
3´-AA cos 3´-G cos GTTAA-5´
lacZ ori lacI CCTAG-5´ ori ori
ori lacZ
M13 M13

Ligation with 30–45 kb Sau 3AI genomic DNA fragment

RS AH
Ampr Ampr
R L R L
5´-AGCTT lacI GAA-3´
3´-AA cos cos GTTAA-5´
lacZ ori ori lacI lacZ ori ori
M13 M13
in vitro packaging

Fig. 4 One approach to the construction of genomic libraries in cosmid vectors (not all
restriction sites are shown): A, AccI; B, BamHI; H, HindIII; R, EcoRI; S, SmaI.

method, this procedure is quick, and rep- fragments make it even more important to
resentative libraries can be obtained from prevent self-ligation of vector fragments.
a small quantity of genomic DNA. Many similar approaches have been sug-
The third ‘‘partial filling-in’’ method, gested and one of them is shown in (Fig. 4)
also avoids fractionation steps (Fig. 3). where the formation of vector-concatemers
Phage arms are prepared as described be- is prevented by partial filling in.
fore (in this particular case SalI and EcoRI A similar effect can be achieved by
are shown, but many other combinations dephosphorylation or by digestion at the
can be used), and the sticky ends produced first step with AccI and SmaI instead of
are partially filled in with the Klenow frag- EcoRI and HindIII. SmaI produces blunt
ment of DNA polymerase I (or other DNA ends and AccI gives sticky ends with only
polymerase) in the presence of dTTP and two protruding base pairs. The ligation of
dCTP. Genomic DNA partially digested these ends will be far less effective than
with Sau3AI is also partially filled in, but that for BamHI and Sau3AI sticky ends
in the presence of dATP and dGTP. Un- (four protruding base pairs).
der such conditions, self-ligation of vector
2.3
arms or genomic DNA is impossible.
Construction of Jumping and Linking
Genomic libraries can be constructed in Libraries. Use of Linking and Jumping
cosmids using the same approaches just Clones to Construct a Physical
described. The absence of selection against Chromosome Map
nonrecombinant vector and the possi-
bility of packaging into phage particles For long-range mapping and cloning of
concatemers composed solely of cosmid large stretches of genomic DNA, the
452 Genomic DNA Libraries, Construction and Applications
two most widely used methods are con-
struction of overlapping DNA sequences
(contigs) using chromosome walking (e.g.
BAC cloning) and chromosome jumping.
The technique of BAC cloning is now used
by many laboratories. Still, this approach
is not devoid of problems and drawbacks.
These problems could be diminished,
however, by using jumping/linking li-
braries. Moreover, jumping and linking
libraries can be used independently for
construction of a long-range restriction
map using pulsed field gel electrophore-
sis (PFGE). Jumping clones contain DNA
sequences adjacent to neighboring NotI
sites, and linking clones contain DNA
sequences surrounding the same restric-
tion site.
The two best-known kinds of jumping
libraries are the NotI jumping library and
the ‘‘general’’ jumping (hopping) library.
The basic principle of both methods is
to clone only the ends of large DNA
fragments rather than continuous DNA
segments, as in BAC clones. Internal
DNA is deleted by controlled biochemical
techniques. The main difference is that in
the first type of library, complete digestion
with a rare cutting enzyme (NotI is the
most popular) is used, and the second
is based on a partial digestion with a
frequently cutting enzyme, followed by
isolation of DNA fragments of desired size.
Using the first type of library, it is possible
to jump over long distances (>1000 kb),
but only from certain starting points
(i.e. those containing the recognition site
for the rare cutting enzyme). Using the
hopping library, it is possible to start
jumping from practically any point and
to cover a defined but shorter distance
(<150 kb). Only the first type of jumping
libraries can be used in conjunction with
linking libraries to create genomic maps,
as described next.
There are two main approaches for
the construction of NotI jumping and
linking libraries. According to the first
method (Fig. 5a), jumping libraries are
constructed as follows: DNA of high
molecular mass, isolated in low-melting
agarose, is completely digested with NotI.
The DNA is ligated at very low concentra-
tion, in the presence of a dephosphorylated
plasmid containing a marker (supF gene),
and is then circularized, trapping the supF
gene, which acts like a marker to select
clones that contain the ends of a long frag-
ment. Another enzyme, one that has no
recognition site in the plasmid, is used
to digest the large circular molecules into
small fragments, each of which is cloned
in a vector phage carrying amber muta-
tions. Recombinant phages containing the
plasmid with the two terminal fragments
are selected in an E. coli strain lacking the
suppressor gene.
The linking library can be constructed
in different ways. In the original protocol,
the genomic DNA is partially digested
with Sau3A and size selected to obtain
10 to 20 kb fragments. The DNA is then
diluted and circularized in the presence
of supF marker plasmid. The circular
products are digested with NotI, ligated
into a NotI-digested suppressor-dependent
vector (NotEMBL3A), and plated on a
suppressor-negative host.
In another approach, DNA from a total
genomic library in a circular form (e.g. cos-
mid) is digested with NotI, and a selectable
marker (e.g. resistance to the antibiotic) is
inserted into recombinants containing this
site. Then these recombinants are selected
by their resistance to the antibiotic.
The most important drawback is that all
these methods used to construct linking
libraries exploit strategies and vectors
different from those used to construct
jumping libraries. Thus, some fragments
 Genomic DNA Libraries, Construction and Applications 453

Genomic DNA

JUMPING LINKING
NotI complete Sau 3A partial digestion
digestion Selection of 20-kb size
DNA fragments
B B
B
B B B Circularization
in presence
of supF marker
B
B B
N
B B

Digest with Bam HI Digest with Not I

Ligate into
B B λ vector N N

(a) λ NM1151ABS λ NotEMBL3A

I. Genomic DNA II. Digest with Bam HI

Digest with Not I

Ligation
Circles Linear

Ligation 5′ GATC 3′
3′ CTAG 5′
Partial filling-in
dATP+ dGTP
5′ GATC GA 3′
Not I 3′AG CTAG 5′
(cannot ligate)

(b) Ligation with λSK4, λSK17, and λSK22 arms

Fig. 5 Two approaches for the construction of jumping and linking libraries: (a) using
supF marker and (b) using partial filling in. (a) Black boxes, supF marker; B, BamHI.
(b) Black and white bars denote NotI sites and vertical slashes represent BamHI sites;
(b I), (b II), construction of the jumping library; (b II), construction of the linking
library. In this case, digestion of the genomic DNA with BamHI is the first step.

present in one library (e.g. jumping) will An integrated approach for construc-
be absent in another (e.g. linking), which tion of jumping and linking libraries is
creates serious problems for the use of outlined in Fig. 5(b). The most important
these libraries in mapping. feature here is that the same vectors and
454 Genomic DNA Libraries, Construction and Applications

protocol are used for construction of both
libraries.
For the linking library, genomic DNA
is completely digested with BamHI. Sub-
sequently, the DNA is self-ligated at a
very low concentration (without a supF
marker) to yield circular molecules as the
main product. To eliminate any linear
molecules, the sticky ends are partly filled
in with the Klenow fragment in the pres-
ence of dATP and dGTP. Since the Klenow
fragment also has exonuclease activity, all
the BamHI sticky ends are neutralized
and nearly all ends generated upon ran-
dom DNA breakage become unavailable
for ligation. Subsequently, the DNA is cut
with NotI and cloned in λSK4, λSK17, and
λSK22 vectors.
The same strategy is applied in the con-
struction of a NotI jumping library. One
initial step is added: genomic DNA is
fully digested with NotI and self-ligated
at a very low concentration. The sub-
sequent steps are the same as for the
linking library. An obvious difference
between this approach and the preced-
ing ones is that the procedure combines
a biochemical selection for NotI jump-
ing fragments with improved ligation
kinetics during the preparation of the li-
braries (see Table 2 for a comparison of
two methods).
In theory, a linking library is sufficient
to construct a physical chromosomal
map. When linking clones are used to
probe a PFGE genomic blot (NotI-digested
genomic DNA), each clone should reveal
two DNA fragments, which are adjacent
in the genome. Thus, in principle, one
should be able to order the rare cutting
sites with just a single library and one
digest, although it will generally not
be possible to distinguish between two
fragments of the same size. To resolve such
ambiguities, it is important to use several
different libraries, each for a particular
enzyme, and to overlap the resulting
patterns just as in ordinary restriction

Tab. 2 Comparison of two main methods for construction of Not I jumping libraries.

Method I (with supF marker) Method II (with partial filling in)

Materials required for construction of jumping library

2 µg genomic DNA (10 mL ligation volume) 1 µg genomic DNA (5 mL ligation volume)
40 µg vector arms 1 µg vector arms
500 µL-sonicated extract (SE) for in vitro 15 µL SE
packaging
2000 µL freeze-thaw extract for in vitro 10 µL FTL
packaging (FTL)
Cloning capacity
0–12 kb 0.2–23 kb
Expected yield
1–5 × 104 /µg genomic DNA 1–5 × 105 /µg genomic DNA
Percentage of recombinants before genetic selection
<1% 45–60%
Maximum sizes of jumps
450 kb >1000 kb

2 3
Not I
1 2 3 4
Chromosomal
DNA
Fig. 6 Long-range mapping using jumping and linking libraries.
fragment analysis. To accomplish this for
the whole human genome will be very
laborious. However, for small stretches
of the genome containing 5 to 10 NotI
sites, this approach can be efficient. The
use of jumping and linking libraries in
a complementary fashion simplifies this
approach (Fig. 6).
Moreover, by cross-screening the two
libraries, it should be possible in prin-
ciple to jump from clone to clone
(–jumping–linking–jumping, etc.), to
generate an ordered map without us-
ing PFGE techniques at all. One of the
main problems with this approach is
that the presence of very CG-rich re-
gions and repeats in the human DNA
may result in cross-hybridization between
different clones.
In the shotgun sequencing approach
for long-range genome mapping, the hy-
bridization technique is replaced by se-
quencing. Jumping and linking clones
are sequenced from the ends and, sub-
sequently, the linear order of the NotI
clones on a chromosome can be estab-
lished using a computer program. Even a
20 bp sequence is likely to uniquely iden-
tify a sequence in the human genome.
The sequence data provide a means to
Genomic DNA Libraries, Construction and Applications 455
Jumping clones
4 5 6 7
Not I Not I Not I
5 6 7 8
Linking clones
discriminate even between different in-
stances of the same class of repeats.
3
Applications and Perspectives
3.1
Cloning DNA Markers Specific for a
Particular Chromosome
It is important, for many purposes, to clone
individual DNA markers for a specific
chromosome. The most straightforward
approach for such purposes is to prepare
special libraries that contain recombinant
clones from a particular chromosome.
One approach is based on the use of the
fluorescence-activated cell sorter (FACS)
system. FACS operates on the principle
of rapid analysis of suspended particles,
single cells, or even chromosomes. A
suspension of chromosomes stained with
fluorescent dyes (usually Hoechst 33258
and chromomycin A3) passes through
the focus of a laser beam that excites
the DNA-bound fluorescent dyes. Equal-
sized droplets are formed by ultrasonic
dispersion, and the droplets containing the
desired chromosome as indicated by the
456 Genomic DNA Libraries, Construction and Applications
fluorescence measurement are deflected
from the main stream by an electric field
and collected in a tube. DNA isolated
from sorted chromosomes can be used
for construction of genomic libraries.
Another approach is based on the use of
hybrid cell lines. To obtain such somatic
cell hybrids, human cells are fused with ro-
dent cells (e.g. mouse or hamster). When
the resulting hybrid cells are grown in cul-
ture, there is a progressive loss of human
chromosomes until only one or a few of
them are left. At this step, some of the seg-
regant hybrid cells can be quite stable. In
a modification of this technique, a human
cell line is transfected with a plasmid vec-
tor or infected with a retroviral vector that
contains a selectable marker (e.g. gpt or
neo). Transfected clones are screened for
those that contain only a single integrated
plasmid per cell (and thus containing a
single marked chromosome). These trans-
formants are micronucleated by prolonged
colcemid treatment and the microcells,
containing only one chromosome, are
produced using special techniques. The
microcells are fused to mouse cells, and
the resulting human–mouse microcell
hybrids, containing the single marked
chromosome, are isolated by growth in
selective medium. Hybrid cells containing
only fragments of human chromosomes
can be produced using human chromo-
somes with translocations and deletions,
or the fragments can be produced ex-
perimentally by X-irradiation (radiation
hybrids). Such hybrid cell lines are very
useful not only for constructing genomic
libraries but also for physical mapping of
already isolated DNA markers and genes.
Another advantage of somatic hybrid cells
is that DNA is available in large amounts
and can be used for different purposes.
Human-specific clones can be isolated
from the library by hybridization to total
human DNA.
At least 40% of the human genome
consists of repetitive sequences of dif-
ferent kinds. Among them, about 50%
are repeats randomly distributed in the
human genome – different kinds of short
(SINE) and long (LINE) interspersed (re-
peating) elements. The most abundant
among them is the Alu repeat family. Alu
repeats are present at >1.0 × 106 copies
per genome with an estimated average
spacing of about 3 kb. Alu repeats have
a length of about 300 bp and consist of
2 homologous units. Related repeats also
exist in other mammals. Different mem-
bers of this family from the same species
usually have homology of 80 to 90% but
are only about 50% identical in different
species. These repeats have conserved and
variable regions. It is possible to find con-
served sequences that are species-specific.
These conserved sequences can be used
as primers for PCR, to specifically am-
plify human sequences in the presence of
nonhuman DNA. These features are the
basis for using Alu repeats for isolation of
human chromosome–specific sequences
from hybrid cell lines containing human
and nonhuman DNA sequences. More-
over, if a hybrid cell line contains only
a short piece of human chromosome,
the Alu-PCR approach can be used for
isolation of markers specific for a de-
fined region of the chromosome. The
principles of the approach are shown
in Fig. 7.
In the case of two hybrid cell lines,
one containing a complete human chro-
mosome (HCL1) and the other carrying
the same chromosome but with a deletion
(HCL2), this method offers the possibil-
ity of obtaining markers specific for the
deletion. This variant can be called the dif-
ferential Alu-PCR approach for obtaining

No PCR amplification
20 kb 1 kb
Human
chromosomal
DNA Alu repeat
Fig. 7 General scheme for Alu-PCR.
DNA markers. The approach is mainly
used in two variants.
In one variant, Alu-PCR is done using
DNA from both cell lines, and the products
of the reactions are separated by agarose
gel electrophoresis. Some bands present
in the products from HCL1 will be absent
among the products from HCL2. These
bands can be excised and cloned, giving
markers localized in the deletion. The
disadvantage of this approach is that
usually such Alu-PCR results in a large
number of products that have a very
complex pattern and look like a smear
on the gel. Among the solutions to this
problem that have been suggested is the
use of more specific primers (only for a
subset of the Alu repeats), or genomic
DNA digested with restriction enzyme.
Another solution is to use hybrid cell lines
that contain only small pieces of human
chromosomes.
The second variant of the Alu-PCR
approach is mainly used in connection
with sources that contain only a limited
amount of human material: YAC clones
and radiation hybrid cell lines containing
small pieces of the human chromosomes.
The YACs can, for example, be used for
Alu-PCR and the total products of the
PCR reaction can be used as a probe to
screen genomic libraries (e.g. in cosmids).
The hybridization pattern reveals which
Genomic DNA Libraries, Construction and Applications 457
12 kb 0.5 kb 0.8 kb 6 kb
ZAlu5
Different Alu-specific
primers ZAlu3
cosmids are present in one YAC, which in
other YACs, and which are present in one
but absent in another. Such an approach
is also useful for mapping.
Another approach to obtaining region-
specific libraries is to use chromosome
microdissection to physically remove the
chromosomal region of interest; the
minute quantities of microdissected DNA
can be subjected to a microcloning proce-
dure. Spreads of human chromosomes are
made and stained using standard cytoge-
netic techniques. DNA from an individual
band is then cut from the chromosome
using ultrafine glass needles or is isolated
with the help of laser equipment. In the
latter case, all other chromosomes are de-
stroyed by the laser, and intact DNA is
present only in the chromosome of in-
terest. DNA obtained from only a few
(2–20) chromosomes is enough for con-
structing a region-specific library. This
DNA is amplified using PCR and cloned
in plasmid or λ vectors.
3.2
CpG Islands as Powerful Markers for
Genome Mapping; CpG Islands and
Functional Genes
Although human DNA is highly methy-
lated, stably unmethylated sequences
(about 1% of the genome) have been
458 Genomic DNA Libraries, Construction and Applications
observed in human chromosomal DNA.
Such sequences occur as discrete ‘‘is-
lands,’’ usually 1 to 2 kb long, that are
dispersed in the genome. They are usu-
ally called CpG (rich) islands because they
contain more than 50% of CG (human
genome contains, on average, about 40%
of CG contents). Their distinctive feature
is the presence of CpG pairs at a pre-
dicted frequency, whereas elsewhere in
the genome, it is present at a frequency
less than 25%. Altogether, there are about
30 000 islands in the haploid genome (the
average spacing is about 1 per 100 kb). It
is now clear that the majority (if not all)
of CpG islands are associated with genes.
It has been shown that recognition sites
for many of the rare cutting enzymes are
closely associated with CpG islands. For
example, at least 82% of all NotI and 76%
of all XmaIII sites are located in the CpG
islands. More than 20% of CpG-island-
containing genes have at least one NotI
site in their sequence, while about 65% of
these genes have XmaIII site(s).
3.3
Alu-PCR and Subtractive Procedures to
Clone CpG Islands from Defined Regions of
Chromosomes
The Alu-PCR approach is used success-
fully for cloning DNA markers, but it
does result in cloning small DNA frag-
ments (500 bp) between Alu sequences.
Alu sequences are distributed in a ran-
dom fashion and are not linked with
genes or other markers. An obvious sug-
gestion for making Alu-PCR more useful
for mapping is to use not simply genomic
DNA from different sources but linking
libraries constructed from these sources.
This modification has certain advantages:
using isolated probes, it is easy to clone
a parental linking clone (e.g. NotI), which
is a natural marker on the chromosome,
convenient for linkage with other markers.
Furthermore, linking clones are located in
CpG-rich islands that are associated with
genes. According to this scheme, linking
libraries are constructed from different hy-
brid cell lines containing either whole or
deleted human chromosomes. Then, total
DNA isolated from these libraries can be
used for Alu-PCR in the manner described
in Sect. 3.1. However, in this case every
PCR product (either discrete bands or total
product) is used as a probe to isolate link-
ing clones from the defined region of the
chromosomes.
Genomic subtractive methods represent
potentially powerful tools for identification
of deleted sequences and cloning re-
gion–specific markers. This approach has
given rewarding results in less-complex
systems such as yeast or cDNA libraries,
but the great complexity of the human
genome has generated serious problems.
These problems can be overcome by reduc-
ing the complexity of the human genomic
sequences. Two approaches have been
suggested to achieve this aim. In one (rep-
resentational difference analysis), only a
subset of genomic sequences (e.g. BamHI
fragments less then 1 kb) is used for
subtractive procedures; this approach will
result in cloning of random sequences. In
the other, NotI linking libraries are used
instead of whole genomic DNA. Interme-
diate products, that is, circles after the
first ligation step can also be successfully
utilized for subtraction (Fig. 8).
The NotI linking library is at least 100
times lower in complexity than the whole
human genome. It is approximately equal
in complexity to the yeast genome. Since
this approach is not linked with Alu
repeats, it offers the possibility of isolating
NotI linking clones that are unavailable for
cloning using Alu-PCR.
 Genomic DNA Libraries, Construction and Applications 459

Normal DNA Tumor DNA

R R R R R R R R R R R

N3 N1 N2 N*3
N1 N2
R R R R RR R R R R R R R R R R
R-digestion

N1 N2 N3 N1 N*3
R R Dilution and R R
R R self ligation R
R

N3 N1 N*3
N1 N2 N-digestion R
R R R R
R N3 R N3

N1 N2 N1 N1 N*3
N1 N2
Not I linker ligation
R R R R

N1 N1 N2 N2 N3 N3 N1 N1
dNTP PCR amplification with dUTP
R R R R

N1 N1 N2 N2 N3 N3 1:100 N1 N1

Denaturation, hybridization

Homohybrids A Heterohybrids Homohybrids B

R R
R

N2 R N2 N1 N1 N1 N1
R R
N3 N3
N1 N1 N1 N1
UDG, mung bean nuclease

N2 R N2 N3 R N3

PCR amplification, labeling, and cloning

Fig. 8 General scheme for the NotI-CODE (Cloning Of DEleted sequences)
procedure. N, NotI sites; R, restriction enzymes recognizing a 4 to 6 bp sequence.
Methylated NotI site is indicated by an asterisk. UDG, uracil-DNA glycosylase
destroys all DNA molecules containing dUTP. Mung bean nuclease digests
double-stranded DNA with mismatches and all single-stranded molecules. Circles
digested with NotI and PCR-amplified are called NR, NotI representation, because
only sequences surrounding NotI sites are present in this amplification product. The
N2 site is deleted and the N*3 site is methylated in tumor DNA.
460 Genomic DNA Libraries, Construction and Applications
3.4
IBD (Identical-by-descent) Fragments for
Identification of Disease Genes
Development of the methods permit-
ting cloning of identical sequences (CIS)
between two sources of DNA can be
very useful for many purposes, including
isolation of disease genes. Identical-by-
descent(IBD) sequences refer to segments
of the human genome shared by two in-
dividuals because they are inherited from
a common ancestor. Regions that are IBD
between individuals affected with a dis-
ease conceivably can contain the disease
gene(s). Two approaches were suggested
to clone such IBD sequences.
In GMS (genomic mismatch scanning),
each DNA preparation is digested with PstI
to yield fragments with protruding 3 ends.
The 3 protruding ends are protected from
digestion by exonuclease III (ExoIII) in
later steps. One of the DNA preparations
is fully methylated at all GATC sites
with E. coli Dam methylase (DAM+).
The other DNA preparation remains
unmethylated. The two DNA pools are
then mixed in equal ratios, denatured,
and allowed to reanneal. Digestion of
the reannealed DNA with both DpnI and
MboI, which cut at fully methylated and
unmethylated GATC sites respectively,
results in digestion of the homohybrids.
The heterohybrids are resistant to both
DpnI and MboI digestion and survive
this treatment. Discrimination between
perfect, mismatch-free heterohybrids and
those with mismatches is done by the
MutHLS enzyme. Only perfect duplexes
will escape nicking during this step.
All DNA molecules, except mismatch-
free ones, are degraded further with
ExoIII. Thus, the full-length, unaltered
heterohybrids are purified from the other
DNA fragments.
Another method was called CIS (cloning
of identical sequences). The scheme of the
CIS-procedure is shown in Fig. 9.
DNA A and B is digested with BamHI
and ligated to special linkers containing
two recognition sites for MvnI. DNA A is
PCR-amplified in the presence of dUTP
and m5dCTP; thus, all cytosines will be
methylated. DNA B is PCR-amplified in
the presence of normal dCTP and biotiny-
lated primers. The two DNA preparations
are mixed in equal ratios, denatured, and
hybridized. Subsequently, the DNA is di-
gested with MvnI. This enzyme can digest
only dsDNA molecules without methylcy-
tosine and will digest all homohybrids B
(they contain at least four sites for MvnI).
The DNA mixture is next treated with
mung bean nuclease. This nuclease de-
stroys all imperfect hybrids and ssDNAs.
Thus, after this treatment we will have only
perfect homohybrids A and perfect (with-
out any mismatches) heterohybrids. The
DNA mixture is then treated with UDG
(uracil-DNA glycosylase). This enzyme re-
moves the uracil base from the DNA and
thus destroys all DNA from individual A.
As a result, there will be only ssDNA from
individual B, which is identical to the DNA
in individual A.
3.5
Strategies to Map and Sequence Genomes;
Hierarchical, Whole-genome, and Slalom
Sequencing Approaches
During the last few years, impressive
progress has been made in mapping and
sequencing whole genomes of various
organisms. Two basic strategies have so far
been employed for genome sequencing.
According to one scheme (hierarchical
approach), the whole genome is mapped
using different types of markers, and a
minimal set of large-insert clones, such
 Genomic DNA Libraries, Construction and Applications 461

DNA A DNA B
x x

B B B B B B

B B Bam HI
Ligate linkers B B B B
B B
B B B B
x x
PCR amplification with
dUTP( ) and dCTP and biotinylated
Bm mB
d5mCTP(m) primers (b) B B
B B b
m Bm mB b
Bm m B b b B B
x x m b b
m
Homohybrids A Heterohybrids Denaturation,
hybridization
Bm mB Bm mB Homohybrids B
x x B B B B
b
m b b b
b
Bm mB B m mB
m b Digest with Mvnl
(destroy homohybrids B)
Bm mB Bm mB
x x B B
m b b
B m mB b
Bm mB
b Mung bean nuclease
m
(destroy imperfect hybrids
Homohybrids A and single-stranded DNA)
Heterohybrids
Bm B Bm mB Bm mB Bm m B
x x
m m b b

UDG (destroy dUTP containing DNA)

b b
Purification of biotinylated
molecules with streptavidin beads,
PCR amplification

Fig. 9 General scheme of the CIS, cloning of identical sequences, procedure. The same
enzymes are used as in the CODE method but in a different order, and the result
is opposite.

as cosmids, PAC, or BAC clones, is fragments of the large-insert clones, are

established. Subsequently, these large- constructed and sequenced.
insert clones are sequenced using a A variant of this strategy, the whole-
shotgun sequencing strategy: small-insert genome shotgun (WGS) sequencing strat-
libraries, containing randomly sheared egy, was developed later and has proved
462 Genomic DNA Libraries, Construction and Applications
valuable. This method involves end se-
quencing of large- (PAC, BAC, cosmids)
and small-insert (2 and 10 kb) clones.
DNA fragments in the small-insert clones
are generated by physical shearing of
whole genomic DNA. All resulting reads
are joined in one sequence with special
computer programs. The WGS method
requires the generation of sequences cov-
ering the whole genome 10 to 15 times.
If sequence coverage is less, then the con-
tig assembly process cannot be completed,
and sequences and clones will just repre-
sent islands, without connection or order.
Therefore, despite impressive technologi-
cal progress, mapping and sequencing of
even small bacterial genomes is expensive
and laborious.
After completion of the genomic se-
quence from one organism, there will
be a great demand, in many cases, for
comparison with the genomes of other in-
dividuals, related species, pathogenic and
nonpathogenic strains, and so on, in the
growing field of comparative genomics.
Such comparisons are highly relevant to
our understanding of human and animal
health, evolution, and ecology.
An efficient strategy for simultaneous
genome mapping and sequencing was re-
cently developed. The approach is based on
slalom libraries, which combine features
of general genomic, jumping, and linking
libraries. First experiments demonstrated
the feasibility of the approach, and showed
that the efficiency (cost-effectiveness and
speed) of existing mapping/sequencing
methods can be improved at least 5- to
10-fold. The slalom allows the establish-
ment of a physical map, with minimal
sets of overlapping clones, which will pin-
point differences in genome organization
between organisms. At the same time, con-
siderable sequence coverage of the genome
(about 50%) will be achieved. This will
make it possible to locate virtually every
gene in a genome, for more detailed (com-
parative) study. Furthermore, since the
efficiency of contig assembly in the slalom
approach is virtually independent of se-
quence read length, even short sequences,
as produced by rapid high-throughput se-
quencing techniques suffice to complete
a physical map and sequence scan of a
small genome. A combination of these new
sequencing techniques with the slalom ap-
proach increases the power of the method
10 to 50 times more and makes it an effi-
cient tool for comparative genomics. The
main principle of the slalom libraries is
shown in Fig. 10.
Two standard genomic EcoRI- and
BamHI-digested libraries are constructed
and they will completely cover the whole
genome. The problem is how to put EcoRI
and BamHI fragments in the correct order.
It can be solved using the connecting
library. The connecting library can be
constructed as follows: DNA isolated ‘‘en
masse’’ from an EcoRI library is digested
with BamHI, circularized in the presence
of the Kanr gene, and plated on agar
with kanamycin. The clones isolated in
this manner will be identical in structure
to the clones from an EcoRI jumping
library prepared in the classical way.
By comparing end sequences in these
three slalom libraries, all clones can be
positioned relative to each other and a
minimal contig of overlapping clones may
be established.
3.6
Restriction-site-tagged Microarrays to Study
CpG-Island Methylation
Methylation, deletions, and amplifications
of cancer genes constitute important
mechanisms in carcinogenesis, and CGI
(CpG-island-containing) microarrays were
 Genomic DNA Libraries, Construction and Applications
Slalom library
1 2
Eco RI
1 2
Connecting
3 4
Contig of
overlapping clones
Eco RI restriction sites
Genomic DNA
Bam HI restriction sites
Bam HI
3 4
Fig. 10 Simplified slalom library approach scheme. Numbers designate
identical end sequences in different libraries that can be joined by a
computer program in a contig of overlapping clones. Dashed lines show
genomic DNA sequences deleted in the connecting library.
suggested to study hypermethylation in
cancer cells. These microarrays can de-
tect methylation changes in tumor DNA.
However, it is unclear whether these mi-
croarrays can be used to detect hemizygous
methylation or copy-number changes. As
the whole human genome DNA is used
for labeling and the clones are small
(0.2–2 kb), this creates a serious prob-
lem. Oligonucleotide-based microarrays
also can be used to study methylation
changes in cancer cells; however, only a
limited number of genes can be tested in
such experiments.
A rough estimation is that the human
genome contains 15 000 to 20 000 NotI
sites. Therefore, thousands of genes could
be tested with NotI clone microarrays. The
fundamental problems for genome-wide
screening using NotI clones are (1) the
463
6 7
6 7
5 8
5 8 9
size and complexity of the human genome,
(2) the number of repeat sequences, and
(3) the comparatively small sizes of the in-
serts in NotI clones (on average 6–8 kb).
A special procedure was developed to am-
plify only regions surrounding NotI sites,
the so-called NotI representations (NRs,
see Fig. 8). Other DNA fragments were
not amplified. Therefore, only 0.1 to 0.5%
of the total DNA is labeled. Interestingly,
sequences surrounding NotI sites contain
10-fold fewer repetitive sequences than the
human genome on average, and there-
fore, these microarrays are not as sensitive
as other methods to the background hy-
bridization caused by repeats. The main
idea of this application is clear from Fig. 8
(tumor DNA). If a particular NotI site is
present in the DNA, then the circle will be
opened with NotI and labeled. However, if
464 Genomic DNA Libraries, Construction and Applications
this NotI site is deleted or methylated, then
the NR will not contain the correspond-
ing DNA sequences. The NotI microarrays
can simultaneously detect copy-number
changes and methylation and, therefore,
they allow the simultaneous study of ge-
netic and epigenetic factors.
The technique underlying the prepa-
ration and use of NotI microarrays is
applicable to any restriction enzyme and
represents a new type of microarray, re-
ferred to as restriction-site-tagged (RST)
microarrays. Such RST microarrays can be
used for different purposes, for example,
to study species composition of complex
microbial systems.
3.7
Restriction-site-tagged Sequences to Study
Biodiversity
There is still much to learn about the
human normal microflora. The human
gut contains approximately 1 to 2 kg
of bacterial cells. The number of these
cells in the intestine is 10 to 100 times
larger than the number of cells in the
human body, but at best 10 to 15%
of the microbial species are known. To
be able to analyze complex microbial
mixtures is of great importance for many
applications. For instance, differences
between individual compositions of the
normal flora will be instrumental for
future analysis of the effects on the
normal flora composition of diet, foods,
geographical location, and medication.
Conversely, the effects of gut microflora on
aging, autoimmunity, and colonic cancer
risk can be studied.
Analysis of human NotI flanking se-
quences (see Sect. 2.3) have demonstrated
that even short sequences surrounding
NotI sites can yield information sufficient
to isolate new genes or uniquely describe
eukaryotic or prokaryotic genomes.
These results led to the realization that
it would be possible to use short se-
quences surrounding NotI sites or, in
general, restriction-site-tagged sequences
(RSTS) for the analysis of complex mi-
crobial mixtures. The collection of NotI
tags represents the NotI sequence passport
or in short NotI passport. NotI passport-
ing means the process of creating NotI
tags/passports.
The general design of the experiment
is as follows. Genomic DNA is digested
with NotI and ligated to a linker with
NotI sticky ends. This linker contains
the BpmI recognition sites. This restric-
tion nuclease cuts 16/14 bp outside of the
recognition site. The ligation mixture is
PCR-amplified with special primers and,
finally, 19 bp tags flanking NotI sites are
generated. DNA from, for instance, fe-
cal samples and surgical specimens is
digested with NotI, and a NotI passport
for the particular specimen is gener-
ated. A comparison of such passports
from different individuals or from the
same individual before and after drug
treatment will reveal the differences be-
tween them.
Analysis of tags for NotI, PmeI, and
SbfI for 70 completely sequenced bacteria
revealed that more than 95% of tags are
species-specific and even different strains
of the same species can be distinguished.
None of these tags matched human or
rodent sequences. Therefore, the approach
allows analysis of complex microbial
mixtures such as those in the human gut
and identification with high sensitivity of a
particular bacterial strain on a quantitative
and qualitative basis.
A similar approach can be used for
eukaryotic cells, for example, for analysis
of cancer cells.
 Genomic DNA Libraries, Construction and Applications
RSTS-passporting and RST-microarray
approaches are mutually complementary.
These two approaches are based on com-
pletely different biochemical techniques
but aim to solve the same problems.
4 Strachan, T., Read, A. (1999) Human Molecular
Summary
While several different strategies are avail-
able to obtain and use DNA markers for
identifying and mapping DNA sequences
in complex organisms, no single system is
likely to suffice for obtaining a complete
and accurate map and sequence of the hu-
man genome. Rather, a combination of
different approaches and vector systems
is needed to corroborate data from differ-
ent sources.
See also Body Expression Map of Primary Literature
Human Genome; Genetics, Molec-
Adorjan, P., Distler, J.,
ular Basis of. Muller, J., Pelet, C.,
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