9.

2 • DNA Cloning by Recombinant DNA Methods 361

■ Functionally significant interactions between proteins can Cutting DNA Molecules into Small Fragments Restriction
be deduced from the phenotypic effects of allele-specific enzymes are endonucleases produced by bacteria that typi-
suppressor mutations or synthetic lethal mutations. cally recognize specific 4- to 8-bp sequences, called restric-
tion sites, and then cleave both DNA strands at this site.
Restriction sites commonly are short palindromic sequences;
9.2 DNA Cloning by Recombinant that is, the restriction-site sequence is the same on each DNA
strand when read in the 5 → 3 direction (Figure 9-10).
DNA Methods For each restriction enzyme, bacteria also produce a
Detailed studies of the structure and function of a gene at the modification enzyme, which protects a bacterium’s own
molecular level require large quantities of the individual gene DNA from cleavage by modifying it at or near each poten-
in pure form. A variety of techniques, often referred to as re- tial cleavage site. The modification enzyme adds a methyl
combinant DNA technology, are used in DNA cloning, which group to one or two bases, usually within the restriction
permits researchers to prepare large numbers of identical site. When a methyl group is present there, the restriction
DNA molecules. Recombinant DNA is simply any DNA mol- endonuclease is prevented from cutting the DNA. Together
ecule composed of sequences derived from different sources. with the restriction endonuclease, the methylating enzyme
The key to cloning a DNA fragment of interest is to link forms a restriction-modification system that protects the
it to a vector DNA molecule, which can replicate within a host DNA while it destroys incoming foreign DNA (e.g.,
host cell. After a single recombinant DNA molecule, com- bacteriophage DNA or DNA taken up during transforma-
posed of a vector plus an inserted DNA fragment, is intro- tion) by cleaving it at all the restriction sites in the DNA.
duced into a host cell, the inserted DNA is replicated along Many restriction enzymes make staggered cuts in the two
with the vector, generating a large number of identical DNA DNA strands at their recognition site, generating fragments
molecules. The basic scheme can be summarized as follows: that have a single-stranded “tail” at both ends (see Figure
9-10). The tails on the fragments generated at a given re-
Vector  DNA fragment striction site are complementary to those on all other frag-
↓ ments generated by the same restriction enzyme. At room
temperature, these single-stranded regions, often called
Recombinant DNA
“sticky ends,” can transiently base-pair with those on other
↓ DNA fragments generated with the same restriction enzyme.
Replication of recombinant DNA within host cells A few restriction enzymes, such as AluI and SmaI, cleave
↓ both DNA strands at the same point within the restriction
site, generating fragments with “blunt” (flush) ends in which
Isolation, sequencing, and manipulation
all the nucleotides at the fragment ends are base-paired to
of purified DNA fragment
nucleotides in the complementary strand.
Although investigators have devised numerous experimen- The DNA isolated from an individual organism has a spe-
tal variations, this flow diagram indicates the essential steps cific sequence, which purely by chance will contain a specific
in DNA cloning. In this section, we cover the steps in this
basic scheme, focusing on the two types of vectors most com-
monly used in E. coli host cells: plasmid vectors, which repli- EcoRI
cate along with their host cells, and bacteriophage  vectors,
which replicate as lytic viruses, killing the host cell and 5 GA AT T C 3
packaging their DNA into virions. We discuss the charac- 3 C T T AAG 5
terization and various uses of cloned DNA fragments in sub-
sequent sections. Cleavage EcoRI

Restriction Enzymes and DNA Ligases Allow

Sticky ends
Insertion of DNA Fragments into Cloning Vectors
A major objective of DNA cloning is to obtain discrete, small
regions of an organism’s DNA that constitute specific genes. 5 G A A T T C 3
3 C T T AA G 5
In addition, only relatively small DNA molecules can be
cloned in any of the available vectors. For these reasons, the ▲ FIGURE 9-10 Cleavage of DNA by the restriction enzyme
very long DNA molecules that compose an organism’s EcoRI. This restriction enzyme from E. coli makes staggered cuts
genome must be cleaved into fragments that can be inserted at the specific 6-bp inverted repeat (palindromic) sequence
into the vector DNA. Two types of enzymes—restriction shown, yielding fragments with single-stranded, complementary
enzymes and DNA ligases—facilitate production of such re- “sticky” ends. Many other restriction enzymes also produce
combinant DNA molecules. fragments with sticky ends.
362 CHAPTER 9 • Molecular Genetic Techniques and Genomics

TABLE 9-1 Selected Restriction Enzymes and Their Recognition Sequences

Enzyme Source Microorganism Recognition Site* Ends Produced

↓
BamHI Bacillus amyloliquefaciens -G-G-A-T-C-C- Sticky
-C-C-T-A-G-G-
↑
↓
EcoRI Escherichia coli -G-A-A-T-T-C- Sticky
-C-T-T-A-A-G-
↑
↓
HindIII Haemophilus influenzae -A-A-G-C-T-T- Sticky
-T-T-C-G-A-A-
↑
↓
KpnI Klebsiella pneumonia -G-G-T-A-C-C- Sticky
-C-C-A-T-G-G-
↑
↓
PstI Providencia stuartii -C-T-G-C-A-G- Sticky
-G-A-C-G-T-C-
↑
↓
SacI Streptomyces achromogenes -G-A-G-C-T-C- Sticky
-C-T-C-G-A-G-
↑
↓
SalI Streptomyces albue -G-T-C-G-A-C- Sticky
-C-A-G-C-T-G-
↑
↓
SmaI Serratia marcescens -C-C-C-G-G-G- Blunt
-G-G-G-C-C-C-
↑
↓
SphI Streptomyces phaeochromogenes -G-C-A-T-G-C- Sticky
-C-G-T-A-C-G-
↑
↓
XbaI Xanthomonas badrii -T-C-T-A-G-A- Sticky
-A-G-A-T-C-T-
↑
*
These recognition sequences are included in a common polylinker sequence (see Figure 9-12).

set of restriction sites. Thus a given restriction enzyme will tor DNA with the aid of DNA ligases. During normal DNA
cut the DNA from a particular source into a reproducible replication, DNA ligase catalyzes the end-to-end joining (lig-
set of fragments called restriction fragments. Restriction en- ation) of short fragments of DNA, called Okazaki fragments.
zymes have been purified from several hundred different For purposes of DNA cloning, purified DNA ligase is used to
species of bacteria, allowing DNA molecules to be cut at a covalently join the ends of a restriction fragment and vector
large number of different sequences corresponding to the DNA that have complementary ends (Figure 9-11). The vec-
recognition sites of these enzymes (Table 9-1). tor DNA and restriction fragment are covalently ligated to-
gether through the standard 3 → 5 phosphodiester bonds
Inserting DNA Fragments into Vectors DNA fragments of DNA. In addition to ligating complementary sticky ends,
with either sticky ends or blunt ends can be inserted into vec- the DNA ligase from bacteriophage T4 can ligate any two
 9.2 • DNA Cloning by Recombinant DNA Methods 363

Genomic DNA fragments tinued propagation of the plasmid through successive gener-
(a) ations of the host cell.
P AATT 3
3
The plasmids most commonly used in recombinant DNA
Vector DNA OH 5 technology are those that replicate in E. coli. Investigators
(a) (b) have engineered these plasmids to optimize their use as vec-
5 OH + P CG 3
tors in DNA cloning. For instance, removal of unneeded por-
3 TTAA P HO 5
tions from naturally occurring E. coli plasmids yields
(c)
P AGCT 3 plasmid vectors, ≈1.2–3 kb in circumferential length, that
HO 5 contain three regions essential for DNA cloning: a replica-
tion origin; a marker that permits selection, usually a drug-
Complementary resistance gene; and a region in which exogenous DNA
ends base-pair fragments can be inserted (Figure 9-12). Host-cell enzymes
replicate a plasmid beginning at the replication origin (ORI),
OH P a specific DNA sequence of 50–100 base pairs. Once DNA
(a) (a) replication is initiated at the ORI, it continues around the cir-
5 AATT 3 Unpaired genomic
+ fragments (b) and (c) cular plasmid regardless of its nucleotide sequence. Thus any
3 T TAA 5 DNA sequence inserted into such a plasmid is replicated
along with the rest of the plasmid DNA.
P HO
Figure 9-13 outlines the general procedure for cloning a
2 ATP DNA fragment using E. coli plasmid vectors. When E. coli
cells are mixed with recombinant vector DNA under certain
T4 DNA ligase
conditions, a small fraction of the cells will take up the plas-
2 AMP + 2 PPi mid DNA, a process known as transformation. Typically,
1 cell in about 10,000 incorporates a single plasmid DNA
(a) (a)
molecule and thus becomes transformed. After plasmid vec-
5 AATT 3 tors are incubated with E. coli, those cells that take up the
3 TTAA 5 plasmid can be easily selected from the much larger number
of cells. For instance, if the plasmid carries a gene that con-
▲ FIGURE 9-11 Ligation of restriction fragments with fers resistance to the antibiotic ampicillin, transformed cells
complementary sticky ends. In this example, vector DNA cut
with EcoRI is mixed with a sample containing restriction
fragments produced by cleaving genomic DNA with several
different restriction enzymes. The short base sequences OR
I
composing the sticky ends of each fragment type are shown. The HindIII
sticky end on the cut vector DNA (a) base-pairs only with the SphI
PstI
complementary sticky ends on the EcoRI fragment (a) in the Region into which
SalI
genomic sample. The adjacent 3-hydroxyl and 5-phosphate XbaI exogenous DNA
groups (red) on the base-paired fragments then are covalently BamHI can be inserted
joined (ligated) by T4 DNA ligase. SmaI
KpnI
SacI r
p
EcoRI am
blunt DNA ends. However, blunt-end ligation is inherently Polylinker Plasmid
inefficient and requires a higher concentration of both DNA cloning vector
and DNA ligase than for ligation of sticky ends.
▲ FIGURE 9-12 Basic components of a plasmid cloning
vector that can replicate within an E. coli cell. Plasmid vectors
E. coli Plasmid Vectors Are Suitable for Cloning contain a selectable gene such as ampr, which encodes the
Isolated DNA Fragments enzyme -lactamase and confers resistance to ampicillin.
Exogenous DNA can be inserted into the bracketed region
Plasmids are circular, double-stranded DNA (dsDNA) mol-
without disturbing the ability of the plasmid to replicate or
ecules that are separate from a cell’s chromosomal DNA. express the ampr gene. Plasmid vectors also contain a replication
These extrachromosomal DNAs, which occur naturally in origin (ORI) sequence where DNA replication is initiated by host-
bacteria and in lower eukaryotic cells (e.g., yeast), exist in a cell enzymes. Inclusion of a synthetic polylinker containing the
parasitic or symbiotic relationship with their host cell. Like recognition sequences for several different restriction enzymes
the host-cell chromosomal DNA, plasmid DNA is duplicated increases the versatility of a plasmid vector. The vector is
before every cell division. During cell division, copies of the designed so that each site in the polylinker is unique on the
plasmid DNA segregate to each daughter cell, assuring con- plasmid.
 364 CHAPTER 9 • Molecular Genetic Techniques and Genomics

can be selected by growing them in an ampicillin-containing

medium.
Plasmid
DNA fragments from a few base pairs up to ≈20 kb com-
vector + DNA fragment
to be cloned monly are inserted into plasmid vectors. If special precautions
r

p
am are taken to avoid manipulations that might mechanically
Enzymatically insert
break DNA, even longer DNA fragments can be inserted into
DNA into plasmid vector a plasmid vector. When a recombinant plasmid with an
inserted DNA fragment transforms an E. coli cell, all the
antibiotic-resistant progeny cells that arise from the initial
Recombinant transformed cell will contain plasmids with the same inserted
plasmid DNA. The inserted DNA is replicated along with the rest of
the plasmid DNA and segregates to daughter cells as the
r

p
am colony grows. In this way, the initial fragment of DNA is
Mix E. coli with plasmids replicated in the colony of cells into a large number of iden-
in presence of CaCl2; heat tical copies. Since all the cells in a colony arise from a single
pulse
transformed parental cell, they constitute a clone of cells, and
Culture on nutrient agar the initial fragment of DNA inserted into the parental plasmid
E. coli plates containing ampicillin
chromosome is referred to as cloned DNA or a DNA clone.
The versatility of an E. coli plasmid vector is increased by
incorporating into it a polylinker, a synthetically generated
sequence containing one copy of several different restriction
sites that are not present elsewhere in the plasmid sequence
Transformed cell Cells that do not (see Figure 9-12). When such a vector is treated with a re-
survives take up plasmid die striction enzyme that recognizes a restriction site in the
on ampicillin plates
polylinker, the vector is cut only once within the polylinker.
Plasmid replication Subsequently any DNA fragment of appropriate length pro-
duced with the same restriction enzyme can be inserted into
the cut plasmid with DNA ligase. Plasmids containing a
polylinker permit a researcher to clone DNA fragments gen-
erated with different restriction enzymes using the same plas-
mid vector, which simplifies experimental procedures.
Cell multiplication
Bacteriophage
Vectors Permit Efficient
Construction of Large DNA Libraries
Vectors constructed from bacteriophage  are about a thou-
sand times more efficient than plasmid vectors in cloning
large numbers of DNA fragments. For this reason, phage 
Technique Animation: Plasmid Cloning

vectors have been widely used to generate DNA libraries,

comprehensive collections of DNA fragments representing
MEDIA CONNECTIONS

the genome or expressed mRNAs of an organism. Two fac-

tors account for the greater efficiency of phage  as a cloning
Colony of cells, each containing copies vector: infection of E. coli host cells by  virions occurs at
of the same recombinant plasmid about a thousandfold greater frequency than transformation
by plasmids, and many more  clones than transformed
▲ EXPERIMENTAL FIGURE 9-13 DNA cloning in a
plasmid vector permits amplification of a DNA fragment.
colonies can be grown and detected on a single culture plate.
A fragment of DNA to be cloned is first inserted into a When a  virion infects an E. coli cell, it can undergo a
plasmid vector containing an ampicillin-resistance gene cycle of lytic growth during which the phage DNA is repli-
(ampr), such as that shown in Figure 9-12. Only the few cated and assembled into more than 100 complete progeny
cells transformed by incorporation of a plasmid molecule phage, which are released when the infected cell lyses (see Fig-
will survive on ampicillin-containing medium. In transformed ure 4-40). If a sample of  phage is placed on a lawn of E. coli
cells, the plasmid DNA replicates and segregates into growing on a petri plate, each virion will infect a single cell.
daughter cells, resulting in formation of an ampicillin- The ensuing rounds of phage growth will give rise to a visi-
resistant colony. ble cleared region, called a plaque, where the cells have been
lysed and phage particles released (see Figure 4-39).
 9.2 • DNA Cloning by Recombinant DNA Methods 365

(a)  Phage genome A  virion consists of a head, which contains the phage
Head Tail Replaceable region Lytic functions
DNA genome, and a tail, which functions in infecting E. coli
host cells. The  genes encoding the head and tail proteins, as
well as various proteins involved in phage DNA replication
and cell lysis, are grouped in discrete regions of the ≈50-kb
0 10 20 30
O
40
P
49 kb viral genome (Figure 9-14a). The central region of the 
Nu1 A J N cro Q genome, however, contains genes that are not essential for
the lytic pathway. Removing this region and replacing it with
(b)  Phage assembly a foreign DNA fragment up to ≈25 kb long yields a recom-
binant DNA that can be packaged in vitro to form phage
capable of replicating and forming plaques on a lawn of E. coli
Preassembled
host cells. In vitro packaging of recombinant  DNA, which
Preassembled  tail mimics the in vivo assembly process, requires preassembled
 head heads and tails as well as two viral proteins (Figure 9-14b).
(49 kb) It is technically feasible to use  phage cloning vectors to
COS COS
generate a genomic library, that is, a collection of  clones
that collectively represent all the DNA sequences in the
Concatomer of  DNA genome of a particular organism. However, such genomic
libraries for higher eukaryotes present certain experimental
Nu1 and A proteins difficulties. First, the genes from such organisms usually con-
promote filling of  head
with DNA between COS tain extensive intron sequences and therefore are too large to
sites be inserted intact into  phage vectors. As a result, the se-
quences of individual genes are broken apart and carried in
more than one  clone (this is also true for plasmid clones).
 genome (1 copy) Moreover, the presence of introns and long intergenic regions
in genomic DNA often makes it difficult to identify the
important parts of a gene that actually encode protein
 tail attaches only sequences. Thus for many studies, cellular mRNAs, which
to filled head
lack the noncoding regions present in genomic DNA, are a
more useful starting material for generating a DNA library.
In this approach, DNA copies of mRNAs, called comple-
mentary DNAs (cDNAs), are synthesized and cloned in
Complete  virion phage vectors. A large collection of the resulting cDNA
clones, representing all the mRNAs expressed in a cell type,
▲ FIGURE 9-14 The bacteriophage
genome and is called a cDNA library.
packaging of bacteriophage
DNA. (a) Simplified map of the 
phage genome. There are about 60 genes in the  genome, only cDNAs Prepared by Reverse Transcription
a few of which are shown in this diagram. Genes encoding of Cellular mRNAs Can Be Cloned
proteins required for assembly of the head and tail are located at
the left end; those encoding additional proteins required for the
to Generate cDNA Libraries
lytic cycle, at the right end. Some regions of the genome can be The first step in preparing a cDNA library is to isolate the
replaced by exogenous DNA (diagonal lines) or deleted (dotted) total mRNA from the cell type or tissue of interest. Because
without affecting the ability of  phage to infect host cells and of their poly(A) tails, mRNAs are easily separated from the
assemble new virions. Up to ≈25 kb of exogenous DNA can be much more prevalent rRNAs and tRNAs present in a cell ex-
stably inserted between the J and N genes. (b) In vivo assembly tract by use of a column to which short strings of thymidyl-
of  virions. Heads and tails are formed from multiple copies of ate (oligo-dTs) are linked to the matrix.
several different  proteins. During the late stage of  infection,
The general procedure for preparing a  phage cDNA li-
long DNA molecules called concatomers are formed; these
brary from a mixture of cellular mRNAs is outlined in Figure
multimeric molecules consist of multiple copies of the 49-kb 
genome linked end to end and separated by COS sites (red),
9-15. The enzyme reverse transcriptase, which is found in
protein-binding nucleotide sequences that occur once in each retroviruses, is used to synthesize a strand of DNA comple-
copy of the  genome. Binding of  head proteins Nu1 and A to mentary to each mRNA molecule, starting from an oligo-dT
COS sites promotes insertion of the DNA segment between two primer (steps 1 and 2 ). The resulting cDNA-mRNA hybrid
adjacent COS sites into an empty head. After the heads are filled molecules are converted in several steps to double-stranded
with DNA, assembled  tails are attached, producing complete  cDNA molecules corresponding to all the mRNA molecules
virions capable of infecting E. coli cells. in the original preparation (steps 3 – 5 ). Each double-stranded
366 CHAPTER 9 • Molecular Genetic Techniques and Genomics

mRNA 5 A A A ....An 3 cDNA contains an oligo-dC
oligo-dG double-stranded re-

3 poly(A) tail
gion at one end and an oligo-dT
oligo-dA double-stranded
1 Hybridize mRNA with region at the other end. Methylation of the cDNA protects
Oligo-dT primer oligo-dT primer
T T T T 5 it from subsequent restriction enzyme cleavage (step 6 ).
To prepare double-stranded cDNAs for cloning, short
AAAA double-stranded DNA molecules containing the recognition
T T T T 5 site for a particular restriction enzyme are ligated to both
2 Transcribe RNA into cDNA ends of the cDNAs using DNA ligase from bacteriophage T4
(Figure 9-15, step 7 ). As noted earlier, this ligase can join
A A A A 3 “blunt-ended” double-stranded DNA molecules lacking
T T T T 5 sticky ends. The resulting molecules are then treated with the
Remove RNA with alkali restriction enzyme specific for the attached linker, generating
3 Add poly(dG) tail cDNA molecules with sticky ends at each end (step 8a ). In
a separate procedure,  DNA first is treated with the same
Single-stranded 3 G G G G T T T T 5 restriction enzyme to produce fragments called  vector
cDNA
Hybridize with arms, which have sticky ends and together contain all the
4
oligo-dC primer genes necessary for lytic growth (step 8b ).
5 C C C C The  arms and the collection of cDNAs, all containing
3 G G G G T T T T 5 complementary sticky ends, then are mixed and joined co-
Synthesize complementary valently by DNA ligase (Figure 9-15, step 9 ). Each of the
5 strand resulting recombinant DNA molecules contains a cDNA lo-
cated between the two arms of the  vector DNA. Virions
Double-stranded 5 C C C C A A A A 3
containing the ligated recombinant DNAs then are assem-
cDNA 3 G G G G T T T T 5
bled in vitro as described above (step 10 ). Only DNA mol-
6 Protect cDNA by ecules of the correct size can be packaged to produce fully
methylation at EcoRI sites infectious recombinant  phage. Finally, the recombinant 
CH3
phages are plated on a lawn of E. coli cells to generate a large
5 C C C C A A A A 3
3 G G G G T T T T 5 number of individual plaques (step 11 ).
CH3

EcoRI linker 7 Ligate cDNA to restriction

GAATTC site linkers
C T T AA G

GAATTC CCCC AAAA GAATTC

C T T AA G GGGG T TTT C T T AA G
Replaceable region
8a Cleave with EcoRI
Bacteriophage λ DNA
AATTC CCCC AAAA G
Cut with EcoRI
G GGGG T TTT CTTAA 8b Remove replaceable region
Sticky end
9 Ligate to λ arms
G AATTC λ vector arms with
CTTAA G sticky ends

10 Package in vitro

Recombinant ▲ EXPERIMENTAL FIGURE 9-15 A cDNA library can

λ virions be constructed using a bacteriophage
vector. A mixture
of mRNAs is the starting point for preparing recombinant 
virions each containing a cDNA. To maximize the size of the
11 Infect E. coli exogenous DNA that can be inserted into the  genome,
Individual the nonessential regions of the  genome (diagonal lines in
λ clones
Figure 9-14) usually are deleted. Plating of the recombinant
phage on a lawn of E. coli generates a set of cDNA clones
representing all the cellular mRNAs. See the text for a step-
by-step discussion.
 9.2 • DNA Cloning by Recombinant DNA Methods 367

Since each plaque arises from a single recombinant

phage, all the progeny  phages that develop are genetically
identical and constitute a clone carrying a cDNA derived Double-
stranded
from a single mRNA; collectively they constitute a  cDNA DNA
library. One feature of cDNA libraries arises because differ-
ent genes are transcribed at very different rates. As a result,
cDNA clones corresponding to rapidly transcribed genes will Melt and place
Bound DNA on filter
be represented many times in a cDNA library, whereas single-
cDNAs corresponding to slowly transcribed genes will be ex- stranded
DNA
tremely rare or not present at all. This property is advanta-
geous if an investigator is interested in a gene that is
transcribed at a high rate in a particular cell type. In this Filter
case, a cDNA library prepared from mRNAs expressed in
that cell type will be enriched in the cDNA of interest, facil-
itating screening of the library for  clones carrying that Incubate with
cDNA. However, to have a reasonable chance of including labeled DNA ( )
clones corresponding to slowly transcribed genes, mam-
malian cDNA libraries must contain 106–107 individual re-
Hybridized
combinant  phage clones. complementary
DNAs
DNA Libraries Can Be Screened by Hybridization
to an Oligonucleotide Probe
Wash away labeled DNA
Both genomic and cDNA libraries of various organisms that does not hybridize
contain hundreds of thousands to upwards of a million in- to DNA bound to filter
dividual clones in the case of higher eukaryotes. Two gen-
eral approaches are available for screening libraries to
identify clones carrying a gene or other DNA region of in-
terest: (1) detection with oligonucleotide probes that bind
to the clone of interest and (2) detection based on expres-
sion of the encoded protein. Here we describe the first
method; an example of the second method is presented in
the next section. Perform autoradiography
The basis for screening with oligonucleotide probes is hy-
bridization, the ability of complementary single-stranded ▲ EXPERIMENTAL FIGURE 9-16 Membrane-hybridization
DNA or RNA molecules to associate (hybridize) specifically assay detects nucleic acids complementary to an
with each other via base pairing. As discussed in Chapter 4, oligonucleotide probe. This assay can be used to detect both
double-stranded (duplex) DNA can be denatured (melted) DNA and RNA, and the radiolabeled complementary probe can
be either DNA or RNA.
into single strands by heating in a dilute salt solution. If the
temperature then is lowered and the ion concentration
raised, complementary single strands will reassociate (hy-
bridize) into duplexes. In a mixture of nucleic acids, only the membrane. Any excess probe that does not hybridize is
complementary single strands (or strands containing com- washed away, and the labeled hybrids are detected by auto-
plementary regions) will reassociate; moreover, the extent of radiography of the filter.
their reassociation is virtually unaffected by the presence of Application of this procedure for screening a  cDNA li-
noncomplementary strands. brary is depicted in Figure 9-17. In this case, a replica of the
In the membrane-hybridization assay outlined in Figure petri dish containing a large number of individual  clones
9-16, a single-stranded nucleic acid probe is used to detect initially is reproduced on the surface of a nitrocellulose mem-
those DNA fragments in a mixture that are complementary brane. The membrane is then assayed using a radiolabeled
to the probe. The DNA sample first is denatured and the sin- probe specific for the recombinant DNA containing the frag-
gle strands attached to a solid support, commonly a nitro- ment of interest. Membrane hybridization with radiolabeled
cellulose filter or treated nylon membrane. The membrane oligonucleotides is most commonly used to screen  cDNA
is then incubated in a solution containing a radioactively la- libraries. Once a cDNA clone encoding a particular protein
beled probe. Under hybridization conditions (near neutral is obtained, the full-length cDNA can be radiolabeled and
pH, 40–65 C, 0.3–0.6 M NaCl), this labeled probe hy- used to probe a genomic library for clones containing frag-
bridizes to any complementary nucleic acid strands bound to ments of the corresponding gene.
368 CHAPTER 9 • Molecular Genetic Techniques and Genomics

Individual phage plaques

5 O Base 1 5 O Base 2
HO DMT O

Master plate of
λ phage plaques 3 3
on E. coli lawn Monomer 1 Monomer 2
O O
Glass
P
support
Place nitrocellulose filter on plate MeO N(IP)2
to pick up phages from each plaque Coupling
(weak acid)

O Base 2
DMT O
Nitrocellulose filter

O
O Base 1
MeO P O
Incubate filter in alkaline
solution to lyse phages and
denature released phage DNA
O

Single-stranded phage
DNA bound to filter Oxidation by I2
Removal of DMT by ZnBr2

Hybridize with labeled probe; O Base 2

HO
perform autoradiography

O
Signal appears over
phage DNA that is O Base 1
MeO P O
complementary
to probe O

▲ EXPERIMENTAL FIGURE 9-17 Phage cDNA libraries can

be screened with a radiolabeled probe to identify a clone of
interest. In the initial plating of a library, the  phage plaques are Repeat process with
not allowed to develop to a visible size so that up to 50,000 monomer 3, monomer 4, etc.
recombinants can be analyzed on a single plate. The appearance
of a spot on the autoradiogram indicates the presence of a Oligonucleotide
recombinant  clone containing DNA complementary to the
probe. The position of the spot on the autoradiogram is the ▲ FIGURE 9-18 Chemical synthesis of oligonucleotides by
mirror image of the position on the original petri dish of that sequential addition of reactive nucleotide derivatives. The first
particular clone. Aligning the autoradiogram with the original petri (3) nucleotide in the sequence (monomer 1) is bound to a glass
dish will locate the corresponding clone from which infectious support by its 3 hydroxyl; its 5 hydroxyl is available for addition
phage particles can be recovered and replated at low density, of the second nucleotide. The second nucleotide in the sequence
resulting in well-separated plaques. Pure isolates eventually are (monomer 2) is derivatized by addition of 4,4-dimethoxytrityl
obtained by repeating the hybridization assay. (DMT) to its 5 hydroxyl, thus blocking this hydroxyl from
reacting; in addition, a highly reactive group (red letters) is
attached to the 3 hydroxyl. When the two monomers are mixed
in the presence of a weak acid, they form a 5 → 3
Oligonucleotide Probes Are Designed Based phosphodiester bond with the phosphorus in the trivalent state.
on Partial Protein Sequences Oxidation of this intermediate increases the phosphorus valency
to 5, and subsequent removal of the DMT group with zinc
Clearly, identification of specific clones by the membrane- bromide (ZnBr2) frees the 5 hydroxyl. Monomer 3 then is added,
hybridization technique depends on the availability of com- and the reactions are repeated. Repetition of this process
plementary radiolabeled probes. For an oligonucleotide to be eventually yields the entire oligonucleotide. Finally, all the methyl
useful as a probe, it must be long enough for its sequence to groups on the phosphates are removed at the same time at
occur uniquely in the clone of interest and not in any other alkaline pH, and the bond linking monomer 1 to the glass
clones. For most purposes, this condition is satisfied by support is cleaved. [See S. L. Beaucage and M. H. Caruthers, 1981,
oligonucleotides containing about 20 nucleotides. This is be- Tetrahedron Lett. 22:1859.]
 9.2 • DNA Cloning by Recombinant DNA Methods 369

cause a specific 20-nucleotide sequence occurs once in every Yeast Genomic Libraries Can Be Constructed
420 (≈1012) nucleotides. Since all genomes are much smaller with Shuttle Vectors and Screened
(≈3  109 nucleotides for humans), a specific 20-nucleotide
sequence in a genome usually occurs only once. Oligonu-
by Functional Complementation
cleotides of this length with a specific sequence can be syn- In some cases a DNA library can be screened for the ability to
thesized chemically and then radiolabeled by using express a functional protein that complements a recessive mu-
polynucleotide kinase to transfer a 32P-labeled phosphate tation. Such a screening strategy would be an efficient way
group from ATP to the 5 end of each oligonucleotide. to isolate a cloned gene that corresponds to an interesting re-
How might an investigator design an oligonucleotide cessive mutation identified in an experimental organism. To
probe to identify a cDNA clone encoding a particular pro- illustrate this method, referred to as functional complementa-
tein? If all or a portion of the amino acid sequence of the pro- tion, we describe how yeast genes cloned in special E. coli
tein is known, then a DNA probe corresponding to a small
region of the gene can be designed based on the genetic code. Polylinker
(a)
However, because the genetic code is degenerate (i.e., many
amino acids are encoded by more than one codon), a probe
ORI
based on an amino acid sequence must include all the possi-
URA3
ble oligonucleotides that could theoretically encode that pep-
tide sequence. Within this mixture of oligonucleotides will be Shuttle vector
one that hybridizes perfectly to the clone of interest.
In recent years, this approach has been simplified by the
availability of the complete genomic sequences for humans ampr
and some important model organisms such as the mouse, ARS
Drosophila, and the roundworm Caenorhabditis elegans.
Using an appropriate computer program, a researcher can CEN
search the genomic sequence database for the coding se-
quence that corresponds to a specific portion of the amino (b)
acid sequence of the protein under study. If a match is found,
then a single, unique DNA probe based on this known ge-
nomic sequence will hybridize perfectly with the clone en-
coding the protein under study. Yeast genomic DNA
Chemical synthesis of single-stranded DNA probes of de- Shuttle vector
Partially digest
fined sequence can be accomplished by the series of reactions Cut with BamHI with Sau3A
shown in Figure 9-18. With automated instruments now
available, researchers can program the synthesis of oligonu-
cleotides of specific sequence up to about 100 nucleotides
long. Alternatively, these probes can be prepared by the poly-
merase chain reaction (PCR), a widely used technique for
amplifying specific DNA sequences that is described later. Ligate

EXPERIMENTAL FIGURE 9-19 Yeast genomic library can

be constructed in a plasmid shuttle vector that can replicate in
yeast and E. coli. (a) Components of a typical plasmid shuttle
vector for cloning Saccharomyces genes. The presence of a yeast
origin of DNA replication (ARS) and a yeast centromere (CEN)
allows, stable replication and segregation in yeast. Also included
is a yeast selectable marker such as URA3, which allows a ura3
Transform E. coli
mutant to grow on medium lacking uracil. Finally, the vector Screen for ampicillin resistance
contains sequences for replication and selection in E. coli (ORI and
ampr) and a polylinker for easy insertion of yeast DNA fragments.
(b) Typical protocol for constructing a yeast genomic library. Partial
digestion of total yeast genomic DNA with Sau3A is adjusted to
generate fragments with an average size of about 10 kb. The
vector is prepared to accept the genomic fragments by digestion Isolate and pool recombinant
with BamHI, which produces the same sticky ends as Sau3A. plasmids from 105 transformed
E. coli colonies
Each transformed clone of E. coli that grows after selection for
ampicillin resistance contains a single type of yeast DNA fragment. Assay yeast genomic library by functional complementation
370 CHAPTER 9 • Molecular Genetic Techniques and Genomics

plasmids can be introduced into mutant yeast cells to iden- which the polylinker has been cleaved with a restriction en-
tify the wild-type gene that is defective in the mutant strain. zyme that produces sticky ends complementary to those on
Libraries constructed for the purpose of screening among the yeast DNA fragments (Figure 9-19b). Because the 10-kb
yeast gene sequences usually are constructed from genomic restriction fragments of yeast DNA are incorporated into the
DNA rather than cDNA. Because Saccharomyces genes do shuttle vectors randomly, at least 105 E. coli colonies, each
not contain multiple introns, they are sufficiently compact so containing a particular recombinant shuttle vector, are nec-
that the entire sequence of a gene can be included in a ge- essary to assure that each region of yeast DNA has a high
nomic DNA fragment inserted into a plasmid vector. To con- probability of being represented in the library at least once.
struct a plasmid genomic library that is to be screened by Figure 9-20 outlines how such a yeast genomic library
functional complementation in yeast cells, the plasmid vector can be screened to isolate the wild-type gene corresponding
must be capable of replication in both E. coli cells and yeast to one of the temperature-sensitive cdc mutations mentioned
cells. This type of vector, capable of propagation in two dif- earlier in this chapter. The starting yeast strain is a double
ferent hosts, is called a shuttle vector. The structure of a typ- mutant that requires uracil for growth due to a ura3
ical yeast shuttle vector is shown in Figure 9-19a (see page mutation and is temperature-sensitive due to a cdc28 muta-
369). This vector contains the basic elements that permit tion identified by its phenotype (see Figure 9-6). Recombi-
cloning of DNA fragments in E. coli. In addition, the shuttle nant plasmids isolated from the yeast genomic library are
vector contains an autonomously replicating sequence (ARS), mixed with yeast cells under conditions that promote trans-
which functions as an origin for DNA replication in yeast; a formation of the cells with foreign DNA. Since transformed
yeast centromere (called CEN), which allows faithful segre- yeast cells carry a plasmid-borne copy of the wild-type
gation of the plasmid during yeast cell division; and a yeast URA3 gene, they can be selected by their ability to grow in
gene encoding an enzyme for uracil synthesis (URA3), which the absence of uracil. Typically, about 20 petri dishes, each
serves as a selectable marker in an appropriate yeast mutant. containing about 500 yeast transformants, are sufficient to
To increase the probability that all regions of the yeast represent the entire yeast genome. This collection of yeast
genome are successfully cloned and represented in the plas- transformants can be maintained at 23 C, a temperature
mid library, the genomic DNA usually is only partially di- permissive for growth of the cdc28 mutant. The entire
gested to yield overlapping restriction fragments of ≈10 kb. collection on 20 plates is then transferred to replica plates,
These fragments are then ligated into the shuttle vector in which are placed at 36 C, a nonpermissive temperature for

Library of yeast genomic DNA

carrying URA3 selective marker

23 °C

Temperature-sensitive
cdc-mutant yeast;
ura3 − (requires uracil) Transform yeast by treatment with
LiOAC, PEG, and heat shock

Plate and incubate at

permissive temperature Only colonies carrying
on medium lacking uracil a wild-type CDC gene
are able to grow
Only colonies
carrying a
URA3 marker
are able to Replica-plate and
grow incubate at nonpermissive
23 °C temperature 36 °C

▲ EXPERIMENTAL FIGURE 9-20 Screening of a yeast are incubated with the mutant yeast cells under conditions
genomic library by functional complementation can that promote transformation. The relatively few transformed
identify clones carrying the normal form of mutant yeast yeast cells, which contain recombinant plasmid DNA, can grow
gene. In this example, a wild-type CDC gene is isolated by in the absence of uracil at 23 C. When transformed yeast
complementation of a cdc yeast mutant. The Saccharomyces colonies are replica-plated and placed at 36 C (a
strain used for screening the yeast library carries ura3 and a nonpermissive temperature), only clones carrying a library
temperature-sensitive cdc mutation. This mutant strain is plasmid that contains the wild-type copy of the CDC gene will
grown and maintained at a permissive temperature (23 C). survive. LiOAC
lithium acetate; PEG
polyethylene glycol.
Pooled recombinant plasmids prepared as shown in Figure 9-19
 9.3 • Characterizing and Using Cloned DNA Fragments 371

cdc mutants. Yeast colonies that carry recombinant plasmids

expressing a wild-type copy of the CDC28 gene will be able
9.3 Characterizing and Using Cloned
to grow at 36 C. Once temperature-resistant yeast colonies DNA Fragments
have been identified, plasmid DNA can be extracted from the
cultured yeast cells and analyzed by subcloning and DNA Now that we have described the basic techniques for using re-
sequencing, topics we take up in the next section. combinant DNA technology to isolate specific DNA clones,
we consider how cloned DNAs are further characterized and
various ways in which they can be used. We begin here with
several widely used general techniques and examine some
KEY CONCEPTS OF SECTION 9.2 more specific applications in the following sections.
DNA Cloning by Recombinant DNA Methods
■ In DNA cloning, recombinant DNA molecules are Gel Electrophoresis Allows Separation of Vector
formed in vitro by inserting DNA fragments into vector DNA from Cloned Fragments
DNA molecules. The recombinant DNA molecules are then
In order to manipulate or sequence a cloned DNA fragment,
introduced into host cells, where they replicate, producing
it first must be separated from the vector DNA. This can be
large numbers of recombinant DNA molecules.
accomplished by cutting the recombinant DNA clone with
■ Restriction enzymes (endonucleases) typically cut DNA the same restriction enzyme used to produce the recombinant
at specific 4- to 8-bp palindromic sequences, producing de- vectors originally. The cloned DNA and vector DNA then
fined fragments that often have self-complementary single- are subjected to gel electrophoresis, a powerful method for
stranded tails (sticky ends). separating DNA molecules of different size.
■ Two restriction fragments with complementary ends can Near neutral pH, DNA molecules carry a large negative
be joined with DNA ligase to form a recombinant DNA charge and therefore move toward the positive electrode dur-
(see Figure 9-11). ing gel electrophoresis. Because the gel matrix restricts ran-
dom diffusion of the molecules, molecules of the same length
■ E. coli cloning vectors are small circular DNA molecules migrate together as a band whose width equals that of the
(plasmids) that include three functional regions: an origin well into which the original DNA mixture was placed at the
of replication, a drug-resistance gene, and a site where a start of the electrophoretic run. Smaller molecules move
DNA fragment can be inserted. Transformed cells carry- through the gel matrix more readily than larger molecules, so
ing a vector grow into colonies on the selection medium that molecules of different length migrate as distinct bands
(see Figure 9-13). (Figure 9-21). DNA molecules composed of up to ≈2000
■ Phage cloning vectors are formed by replacing nonessen- nucleotides usually are separated electrophoretically on
tial parts of the  genome with DNA fragments up to polyacrylamide gels, and molecules from about 200 nu-
≈25 kb in length and packaging the resulting recombinant cleotides to more than 20 kb on agarose gels.
DNAs with preassembled heads and tails in vitro. A common method for visualizing separated DNA bands
on a gel is to incubate the gel in a solution containing the
■ In cDNA cloning, expressed mRNAs are reverse-
fluorescent dye ethidium bromide. This planar molecule
transcribed into complementary DNAs, or cDNAs. By a
binds to DNA by intercalating between the base pairs. Bind-
series of reactions, single-stranded cDNAs are converted
ing concentrates ethidium in the DNA and also increases its
into double-stranded DNAs, which can then be ligated into
intrinsic fluorescence. As a result, when the gel is illuminated
a  phage vector (see Figure 9-15).
with ultraviolet light, the regions of the gel containing DNA
■ A cDNA library is a set of cDNA clones prepared from fluoresce much more brightly than the regions of the gel
the mRNAs isolated from a particular type of tissue. A without DNA.
genomic library is a set of clones carrying restriction frag- Once a cloned DNA fragment, especially a long one, has
ments produced by cleavage of the entire genome. been separated from vector DNA, it often is treated with var-
■ The number of clones in a cDNA or genomic library ious restriction enzymes to yield smaller fragments. After sep-
must be large enough so that all or nearly all of the orig- aration by gel electrophoresis, all or some of these smaller
inal nucleotide sequences are present in at least one clone. fragments can be ligated individually into a plasmid vector
and cloned in E. coli by the usual procedure. This process,
■ A particular cloned DNA fragment within a library can known as subcloning, is an important step in rearranging
be detected by hybridization to a radiolabeled oligonu- parts of genes into useful new configurations. For instance, an
cleotide whose sequence is complementary to a portion of investigator who wants to change the conditions under which
the fragment (see Figures 9-16 and 9-17). a gene is expressed might use subcloning to replace the nor-
■ Shuttle vectors that replicate in both yeast and E. coli mal promoter associated with a cloned gene with a DNA seg-
can be used to construct a yeast genomic library. Specific ment containing a different promoter. Subcloning also can be
genes can be isolated by their ability to complement the cor- used to obtain cloned DNA fragments that are of an appro-
responding mutant genes in yeast cells (see Figure 9-20). priate length for determining the nucleotide sequence.
372 CHAPTER 9 • Molecular Genetic Techniques and Genomics

DNA restriction fragments  EXPERIMENTAL FIGURE 9-21 Gel electrophoresis

separates DNA molecules of different lengths. A gel is
prepared by pouring a liquid containing either melted agarose
or unpolymerized acrylamide between two glass plates a few
millimeters apart. As the agarose solidifies or the acrylamide
polymerizes into polyacrylamide, a gel matrix (orange ovals) forms
consisting of long, tangled chains of polymers. The dimensions of
the interconnecting channels, or pores, depend on the
concentration of the agarose or acrylamide used to form the gel.
Place mixture in the well of The separated bands can be visualized by autoradiography (if the
an agarose or polyacrylamide fragments are radiolabeled) or by addition of a fluorescent dye
gel. Apply electric field (e.g., ethidium bromide) that binds to DNA.

Well
– Cloned DNA Molecules Are Sequenced Rapidly
Gel particle by the Dideoxy Chain-Termination Method
The complete characterization of any cloned DNA fragment
requires determination of its nucleotide sequence. F. Sanger
and his colleagues developed the method now most commonly
used to determine the exact nucleotide sequence of DNA frag-
Pores
ments up to ≈500 nucleotides long. The basic idea behind this
method is to synthesize from the DNA fragment to be se-
quenced a set of daughter strands that are labeled at one end
and differ in length by one nucleotide. Separation of the trun-
+ cated daughter strands by gel electrophoresis can then estab-
lish the nucleotide sequence of the original DNA fragment.
Molecules move through pores
in gel at a rate inversely Synthesis of truncated daughter stands is accomplished by
proportional to their chain length use of 2,3-dideoxyribonucleoside triphosphates (ddNTPs).
These molecules, in contrast to normal deoxyribonucleotides
– (dNTPs), lack a 3 hydroxyl group (Figure 9-22). Although
ddNTPs can be incorporated into a growing DNA chain by

 
O O
 
O P O O P O

O O
 
O P O O P O

O O
+  
O P O O P O
Subject to autoradiography Base Base
O O
or incubate with fluorescent dye
CH2 CH2
O O
H H H H
H H H H
3' 3'
Signal corresponding OH H H H
to DNA band
Deoxyribonucleoside Dideoxyribonucleoside
triphosphate triphosphate
(dNTP) (ddNTP)

▲ FIGURE 9-22 Structures of deoxyribonucleoside

triphosphate (dNTP) and dideoxyribonucleoside triphosphate
(ddNTP). Incorporation of a ddNTP residue into a growing DNA
strand terminates elongation at that point.
 9.3 • Characterizing and Using Cloned DNA Fragments 373

(b)

Technique Animation: Dideoxy Sequencing of DNA

MEDIA CONNECTIONS
Primer 5
Template 3 5
DNA polymerase
+ dNTPs (100 µM)
(a)
5 T A G C T G A C T C 3
3 A T C G A C T G A G T C A A G A A C T A T T GGG C T T A A . . .

DNA polymerase + ddATP + ddGTP + ddTTP + ddCTP

+ dATP, dGTP, dCTP, dTTP (1 µM) (1 µM) (1 µM) (1 µM)
+ ddGTP in low concentration

5 T A G C T G A C T C A G 3 A G T C
3 A T C G A C T G A G T C A A G A A C T A T T GGG C T T A A . . .
+
5 T A G C T G A C T C A G T T C T T G 3 A G T C
3 A T C G A C T G A G T C A A G A A C T A T T GGG C T T A A . . .
+
A G T C
5 T A G C T G A C T C A G T T C T T G A T A A C C C G 3
3 A T C G A C T G A G T C A A G A A C T A T T GGG C T T A A . . . etc.
etc. etc. etc.

(c) Denature and separate

Denature daughter
and separate strands
daughter by electrophoresis
strands by electrophoresis

T NNNN AA T G CCAAT ACG ACT CACT A T AG G G C G A AT T CG A G C T C G G T AC C C G GG G A T C C T C T A G A G T C G A C C T G C A G G C A T G C A A G C T T G A G T A T T C T

10 20 30 40 50 60 70 80 90

AT A GT G T CAC C T A A A T AG CT TG GCG T A A T C AT GG T C A T A G C TG T T TC C TG TG TG A A AT T G T T A T C C G C T C A C A A T T C CAC A C A A C A T A

100 110 120 130 140 150 160 170 180

▲ EXPERIMENTAL FIGURE 9-23 Cloned DNAs can be (truncated) daughter fragments ending at every occurrence of
sequenced by the Sanger method, using fluorescent- ddGTP. (b) To obtain the complete sequence of a template
tagged dideoxyribonucleoside triphosphates (ddNTPs). (a) DNA, four separate reactions are performed, each with a
A single (template) strand of the DNA to be sequenced (blue different dideoxyribonucleoside triphosphate (ddNTP). The
letters) is hybridized to a synthetic deoxyribonucleotide primer ddNTP that terminates each truncated fragment can be
(black letters). The primer is elongated in a reaction mixture identified by use of ddNTPs tagged with four different
containing the four normal deoxyribonucleoside triphosphates fluorescent dyes (indicated by colored highlights). (c) In an
plus a relatively small amount of one of the four automated sequencing machine, the four reaction mixtures are
dideoxyribonucleoside triphosphates. In this example, ddGTP subjected to gel electrophoresis and the order of appearance
(yellow) is present. Because of the relatively low of each of the four different fluorescent dyes at the end of the
concentration of ddGTP, incorporation of a ddGTP, and thus gel is recorded. Shown here is a sample printout from an
chain termination, occurs at a given position in the sequence automated sequencer from which the sequence of the original
only about 1 percent of the time. Eventually the reaction template DNA can be read directly. N
nucleotide that
mixture will contain a mixture of prematurely terminated cannot be assigned. [Part (c) from Griffiths et al., Figure 14-27.]
 374 CHAPTER 9 • Molecular Genetic Techniques and Genomics

DNA polymerase, once incorporated they cannot form a tration of one of the four ddNTPs in addition to higher con-
phosphodiester bond with the next incoming nucleotide centrations of the normal dNTPs. In each reaction, the ddNTP
triphosphate. Thus incorporation of a ddNTP terminates is randomly incorporated at the positions of the corresponding
chain synthesis, resulting in a truncated daughter strand. dNTP, causing termination of polymerization at those posi-
Sequencing using the Sanger dideoxy chain-termination tions in the sequence (Figure 9-23a). Inclusion of fluorescent
method begins by denaturing a double-stranded DNA frag- tags of different colors on each of the ddNTPs allows each set
ment to generate template strands for in vitro DNA synthesis. of truncated daughter fragments to be distinguished by their
A synthetic oligodeoxynucleotide is used as the primer for four corresponding fluorescent label (Figure 9-23b). For example,
separate polymerization reactions, each with a low concen- all truncated fragments that end with a G would fluoresce one
color (e.g., yellow), and those ending with an A would fluo-
resce another color (e.g., red), regardless of their lengths. The
mixtures of truncated daughter fragments from each of the
Denaturation of DNA four reactions are subjected to electrophoresis on special poly-
Cycle 1
Annealing of primers
acrylamide gels that can separate single-stranded DNA mole-
cules differing in length by only 1 nucleotide. In automated
DNA sequencing machines, a fluorescence detector that can
distinguish the four fluorescent tags is located at the end of the
Elongation of primers
gel. The sequence of the original DNA template strand can be
determined from the order in which different labeled frag-
ments migrate past the fluorescence detector (Figure 9-23c).
Denaturation of DNA In order to sequence a long continuous region of genomic
Cycle 2 DNA, researchers often start with a collection of cloned
Annealing of primers
DNA fragments whose sequences overlap. Once the se-
quence of one of these fragments is determined, oligonu-
cleotides based on that sequence can be chemically
synthesized for use as primers in sequencing the adjacent
overlapping fragments. In this way, the sequence of a long
Elongation of primers stretch of DNA is determined incrementally by sequencing of
the overlapping cloned DNA fragments that compose it.

 EXPERIMENTAL FIGURE 9-24 The polymerase chain

reaction (PCR) is widely used to amplify DNA regions of
known sequences. To amplify a specific region of DNA, an
Denaturation of DNA investigator will chemically synthesize two different
Cycle 3
Annealing of primers oligonucleotide primers complementary to sequences of
Technique Animation: Polymerase Chain Reaction

approximately 18 bases flanking the region of interest (designated

as light blue and dark blue bars). The complete reaction is
composed of a complex mixture of double-stranded DNA (usually
genomic DNA containing the target sequence of interest), a
stoichiometric excess of both primers, the four deoxynucleoside
triphosphates, and a heat-stable DNA polymerase known as Taq
polymerase. During each PCR cycle, the reaction mixture is first
MEDIA CONNECTIONS

heated to separate the strands and then cooled to allow the

primers to bind to complementary sequences flanking the region
Elongation of primers to be amplified. Taq polymerase then extends each primer from
its 3 end, generating newly synthesized strands that extend in
the 3 direction to the 5 end of the template strand. During the
third cycle, two double-stranded DNA molecules are generated
equal in length to the sequence of the region to be amplified. In
each successive cycle the target segment, which will anneal to
the primers, is duplicated, and will eventually vastly outnumber all
other DNA segments in the reaction mixture. Successive PCR
cycles can be automated by cycling the reaction for timed
intervals at high temperature for DNA melting and at a defined
lower temperature for the annealing and elongation portions of
the cycle. A reaction that cycles 20 times will amplify the specific
Cycles 4, 5, 6, etc. target sequence 1-million-fold.
 9.3 • Characterizing and Using Cloned DNA Fragments 375

The Polymerase Chain Reaction Amplifies a target sequence for about 20 PCR cycles, cleavage with the
Specific DNA Sequence from a Complex Mixture appropriate restriction enzymes produces sticky ends that
allow efficient ligation of the fragment into a plasmid vec-
If the nucleotide sequences at the ends of a particular DNA tor cleaved by the same restriction enzymes in the
region are known, the intervening fragment can be ampli- polylinker. The resulting recombinant plasmids, all carrying
fied directly by the polymerase chain reaction (PCR). Here the identical genomic DNA segment, can then be cloned in
we describe the basic PCR technique and three situations in
which it is used.
The PCR depends on the ability to alternately denature
Region to be amplified
(melt) double-stranded DNA molecules and renature (an-
neal) complementary single strands in a controlled fashion. 5 3
As in the membrane-hybridization assay described earlier, 3 5
the presence of noncomplementary strands in a mixture has 5 G G A T C C 3
DNA synthesis
little effect on the base pairing of complementary single DNA Primer 1
Round 1
strands or complementary regions of strands. The second re-
3 5
quirement for PCR is the ability to synthesize oligonu-
cleotides at least 18–20 nucleotides long with a defined 5 G G A T C C 3
3 T T C G A A 5
sequence. Such synthetic nucleotides can be readily produced DNA synthesis
Primer 2
with automated instruments based on the standard reaction Round 2
scheme shown in Figure 9-18. 5 G G A T C C 3
As outlined in Figure 9-24, a typical PCR procedure be-
3 C C T A G G T T C G A A 5
gins by heat-denaturation of a DNA sample into single 5 G G A T C C 3
DNA synthesis
strands. Next, two synthetic oligonucleotides complemen- Primer 1
Round 3
tary to the 3 ends of the target DNA segment of interest are
added in great excess to the denatured DNA, and the tem- 3 C C T A G G T T C G A A 5
perature is lowered to 50–60 C. These specific oligonu- 5 G G A T C C A A G C T T 3

cleotides, which are at a very high concentration, will BamHI site HindIII site
hybridize with their complementary sequences in the DNA Continue for ~20
sample, whereas the long strands of the sample DNA remain PCR cycles
apart because of their low concentration. The hybridized Cut with restriction
enzymes
oligonucleotides then serve as primers for DNA chain syn-
thesis in the presence of deoxynucleotides (dNTPs) and a 3 G T T C G A 5
5 G A T C C A 3
temperature-resistant DNA polymerase such as that from
Sticky end
Thermus aquaticus (a bacterium that lives in hot springs). Sticky end

This enzyme, called Taq polymerase, can remain active even Ligate with plasmid vector
with sticky ends
after being heated to 95 C and can extend the primers at
temperatures up to 72 C. When synthesis is complete, the
whole mixture is then heated to 95 C to melt the newly
formed DNA duplexes. After the temperature is lowered
again, another cycle of synthesis takes place because excess
primer is still present. Repeated cycles of melting (heating)
and synthesis (cooling) quickly amplify the sequence of in-
terest. At each cycle, the number of copies of the sequence
between the primer sites is doubled; therefore, the desired se-
quence increases exponentially—about a million-fold after
20 cycles—whereas all other sequences in the original DNA ▲ EXPERIMENTAL FIGURE 9-25 A specific target region
sample remain unamplified. in total genomic DNA can be amplified by PCR for use in
cloning. Each primer for PCR is complementary to one end of
the target sequence and includes the recognition sequence for a
Direct Isolation of a Specific Segment of Genomic DNA
restriction enzyme that does not have a site within the target
For organisms in which all or most of the genome has been
region. In this example, primer 1 contains a BamHI sequence,
sequenced, PCR amplification starting with the total ge- whereas primer 2 contains a HindIII sequence. (Note that for
nomic DNA often is the easiest way to obtain a specific clarity, in any round, amplification of only one of the two strands
DNA region of interest for cloning. In this application, the is shown, the one in brackets.) After amplification, the target
two oligonucleotide primers are designed to hybridize to se- segments are treated with appropriate restriction enzymes,
quences flanking the genomic region of interest and to in- generating fragments with sticky ends. These can be
clude sequences that are recognized by specific restriction incorporated into complementary plasmid vectors and cloned in
enzymes (Figure 9-25). After amplification of the desired E. coli by the usual procedure (see Figure 9-13).
376 CHAPTER 9 • Molecular Genetic Techniques and Genomics

E. coli cells. With certain refinements of the PCR, DNA pler method for identifying genes associated with a particu-
segments 10 kb in length can be amplified and cloned in lar mutant phenotype than screening of a library by func-
this way. tional complementation (see Figure 9-20).
Note that this method does not involve cloning of large The key to this use of PCR is the ability to produce mu-
numbers of restriction fragments derived from genomic tations by insertion of a known DNA sequence into the
DNA and their subsequent screening to identify the specific genome of an experimental organism. Such insertion muta-
fragment of interest. In effect, the PCR method inverts this tions can be generated by use of mobile DNA elements,
traditional approach and thus avoids its most tedious as- which can move (or transpose) from one chromosomal site
pects. The PCR method is useful for isolating gene sequences to another. As discussed in more detail in Chapter 10, these
to be manipulated in a variety of useful ways described later. DNA sequences occur naturally in the genomes of most or-
In addition the PCR method can be used to isolate gene se- ganisms and may give rise to loss-of-function mutations if
quences from mutant organisms to determine how they dif- they transpose into a protein-coding region.
fer from the wild-type. For example, researchers have modified a Drosophila mo-
bile DNA element, known as the P element, to optimize its
Preparation of Probes Earlier we discussed how oligonu- use in the experimental generation of insertion mutations.
cleotide probes for hybridization assays can be chemically Once it has been demonstrated that insertion of a P element
synthesized. Preparation of such probes by PCR amplifica- causes a mutation with an interesting phenotype, the genomic
tion requires chemical synthesis of only two relatively short sequences adjacent to the insertion site can be amplified by a
primers corresponding to the two ends of the target se- variation of the standard PCR protocol that uses synthetic
quence. The starting sample for PCR amplification of the tar- primers complementary to the known P-element sequence but
get sequence can be a preparation of genomic DNA. that allows unknown neighboring sequences to be amplified.
Alternatively, if the target sequence corresponds to a mature Again, this approach avoids the cloning of large numbers of
mRNA sequence, a complete set of cellular cDNAs synthe- DNA fragments and their screening to detect a cloned DNA
sized from the total cellular mRNA using reverse transcrip- corresponding to a mutated gene of interest.
tase or obtained by pooling cDNA from all the clones in a  Similar methods have been applied to other organisms
cDNA library can be used as a source of template DNA. To for which insertion mutations can be generated using either
generate a radiolabeled product from PCR, 32P-labeled mobile DNA elements or viruses with sequenced genomes
dNTPs are included during the last several amplification cy- that can insert randomly into the genome.
cles. Because probes prepared by PCR are relatively long and
have many radioactive 32P atoms incorporated into them,
these probes usually give a stronger and more specific signal Blotting Techniques Permit Detection of Specific
than chemically synthesized probes. DNA Fragments and mRNAs with DNA Probes
Two very sensitive methods for detecting a particular DNA
Tagging of Genes by Insertion Mutations Another useful or RNA sequence within a complex mixture combine sepa-
application of the PCR is to amplify a “tagged” gene from ration by gel electrophoresis and hybridization with a com-
the genomic DNA of a mutant strain. This approach is a sim- plementary radiolabeled DNA probe. We will encounter

DNA

Cleave with
restriction enzymes
Gel Nitrocellulose Autoradiogram
Electrophoresis

Filter Nitrocellulose Hybridize with

paper Gel labeled DNA or
RNA probe

Alkaline solution

Capillary action transfers

DNA from gel to nitrocellulose

▲ EXPERIMENTAL FIGURE 9-26 Southern blot technique hybridize to a labeled probe will give a signal on an
can detect a specific DNA fragment in a complex mixture of autoradiogram. A similar technique called Northern blotting
restriction fragments. The diagram depicts three different detects specific mRNAs within a mixture. [See E. M. Southern,
restriction fragments in the gel, but the procedure can be applied 1975, J. Mol. Biol. 98:508.]
to a mixture of millions of DNA fragments. Only fragments that