Introduction

14 i/ham

to recombinant-DNA

technology12

L Carroll
Recombinant-DNA in virtually brief to to the along cloning with the gene used every identify principles a description these large aspect genes, of this amplify exposition if necessary. technology of the is to to provide isolate and of an genes. mm to the clone very approach J (liii is now biological an cornDNA that make either up a mammalian DNA strand directions the The and on chromosome. thus individual a chromosome. Genes genes One may he may gene

ABSTRACl
rnonlv The the terest. short gating used purpose approaches to introduction and

sciences. located on outline of be oriented gene

in opposite

of incodes for a single genes. isA illustrated in genes (mRNA) (gene

polypeptide: Figure 2. transcription)

flow synthesis

of genetic information of messenger RNA by genetic ofthe RNA elements gene. These polymerase core secontrol.

ofseparating

large to
Niur

is controlled portion enzyme

is provided.

propa- that usually lie 5 to the ,)ro,flokr elements serve to the quences These

protein-coding to focus the

I 993:58(suppl):249S-58S.

KEY
RNA chain

WORDS
polvrnerase. reaction. \east

Plasmid,
cloning. artificial

bacteriophage. reverse
restriction endonuclease. chromosome

transcriptase. polymerase

transcription start point. Many promoters share that are essential for effective transcription core sequences (bo.ves) include the thymine-adenine-thy(TATA) box, and box. the cytosine-adenine-adenine-thythe guanine-cytosine (GC) box. upstream(5) whereas start point for in exons.

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mine-adenine mine (CAAT)

The mRNA regions

Introduction

CAAT and GC boxes the TATA box seems synthesis. The genes

are located further to control the actual ofeukaryotes are

arranged

of the gene represented in the mature mRNA. Exons are sepaThe overall goal of recombinant-DNA technology is to idenrated by intervening sequences or introns. which are spliced from tify, isolate. manipulate. and re-express genes from a given host RNA template during RNA processing. Other eu( I -9). Some of the practical goals of such cut-and-paste tech-the primary karyotic-mRNA processing events include the addition of a nology is to 1) develop a basic understanding of the function methylated guanine residue connected by a 5-5 triphosphate and regulation of known gene products. 2) identify new genes to the first nucleotide ofthe mRNA (cap site) as well as whose protein products have not been isolated (reverse genetics). linkage the addition of a stretch of polyadenylic acid residues at the 3 3) correct endogenous genetic defects (eg. sickle cell anemia). 4) terminus. The mRNA is translated into protein with a methiexpress foreign genes in disease-susceptible hosts (eg. diseaseserving as a translation start site. Proteins also have polarity resistance genes in agricultural crops). and 5) manufacture large onine with an unpaired amino group (NH2) at one end and a free quantities ofa protein product for widespread use (eg. antibodies carhoxyl group at the other. The amino-terminal part of the in tobacco plants). Any discussion ofthis methodology should begin ecule by with itself. hydrogen a description DNA bonds ofthe between unique oftwo their features antiparallel nitrogenous-base ofthe DNA side mol-protein bound is encoded by the 5 end ofthe mRNA. is composed strands

chains. Restriction endonucleases Genetic information is provided by the purine and pvrimidine The first breakthrough in recombinant bases. which are linked to a sugar phosphate backbone (Fig). 1 of enzymes capable This structural support is a series of deoxyribose residues linked the identification reproducible fragments. b phosphodiester bonds, which are created by covalently joining into discrete a hydroxvl group at the 3-carbon residue with a phosphate group adjacent sugar group. Thus DNA unpaired 5 phosphate other. By convention, of the gene oriented

of

DNA cleaving

technology duplex

was DNA

These resiric!iomz

endomzu-

c/cases have now been identified in many prokaryotes. and they position on one deoxyribose recognize and cleave both strands of DNA at well-defined base located on the 5 carbon of an sequences [usually composed of 4, 6. or 8 base pairs (bp)]. Most strands have polarity with an of cleavage sites are characterized by an axis of symmetry at one end and a 3 hvdroxyl group at the these may take place symmetrically on either side gene sequences are written with the 5 end(Fig 3). Cleavage to the left and the 3 end to the right

(Fig

2).
From the Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, and the Division of Hematology/Oncology. St Louis Childrens Hospital. St Louis. 2 Address reprint requests to WL Carroll, Program in Molecular and over Biology and Genetics. University of Utah. Building 533. Room 3260. strands of Salt Lake City. UT 84112.

Gene
The a 1000

structure
gene forms the genes may basic unit of genetic on information

he located

the uninterrupted

.ini

I (Ii,,

.Vuir

1993:58(suppl):249S-58S.

Printed

in

USA.

1993

American

Society

for Clinical

Nutrition

249S

250S

CARROLL
step by serting which hosts, (Fig 4). digesting the allow usually

In its simplest
host fragments successful bacteria. method. DNA One fragments in DNA into with

form
any propagation

genecloning
one of four of the

is accomplished endonucleases basic vector gene a general systems. in vectors cloning which features an copy (eg. that have alternative example used vehicles replicate of origin of number antibiotic incorBoth of to and in-

restriction

Figure of

5 illustrates the most is plasmids. DNA bacteria.

one such propagate are circular

common These molecules, Common

double-stranded many to used laboratory the ability cell. allowing a gene

extrachromosomally currently replication. per bacterial resistance) porated a large

plasmid vectors be maintained that confers of host

include in high

a phenotype bacteria

selection

the plasmid. and number of restriction

a cloning (po/iIinker) site containing endonuclease cleavage sites. are digested molecules vectors of restriction with with contain endonuclease

the vector and tical restriction ible linker ends. sequences

the DNA to be cloned endonuclease, yielding commercial with a large plasmid number

an idencompatpoly-

Because

cleavage sites, these vectors will conveniently erated by digestion with many enzymes. The of vector and insert are able to anneal to complementary covalent bonds a closed. The been circular plasmid rendered base between is then competent pairing. DNA incubated to take

accept DNA genoverhanging termini one another through ligase forms resulting in that the agar have

Downloaded from www.ajcn.org by guest on May 31, 2011

The enzyme DNA strands at these sites, plasmid. with up exogenous bacterial DNA

recombinant

cells

by a variety plasmid plate con-

of methods. Bacterial cells that have incorporated can be selected for by plating the reaction on an
5endW

------..-.-.----.-..

--

taming an susceptible

antibiotic to the

(eg, ampicillin). antibiotic. and

only

The bacterial those cells

host is usually that have taken

FIG 1. The DNA molecule. DNA is composed twoof antiparallel strands hound together through hydrogen bonding between complementary nucleotides [adenine-thymine. guanine-cytosine (A-T. G-C)J on the opposite strands. The sugar phosphate backbone ofan individual strand is linked together through phosphodiester bonds between a phosphate group on the 5 carbon ofone residue and a hydroxyl group attached to the 3 carbon ofthe adjacent sugar. Reproduced with permission from J Darnell. H Lodish. and D Baltimore (10).

up the plasmid acquire the gene that codes for antibiotic resistance. This form of selection detects only bacteria that can take up circular plasmids. It does not distinguish plasmids with inserted DNA (recombinants) lacking inserts. Recently. plasmids been can these sidase tional venient easily vectors gene be identified carry coding by from constructed histochemical recircularized such that vector recombinants Typically. funcDNA

techniques.

a portion of the Escherichia for the NH2-terminal truncated that does

co/i (1-galactosequence of the

of the (sticky

termini frame but only adds a few harmless amino acids to the shortened along the 13-galactosidase protein. When these vectors are inserted into axis itself leaving termini with no single stranded overhang host bacterial strains that code for the carboxy-terminal portion (blunt ends) (Table 1). Restriction endonucleases are named of(3-galactosidase. both fragments can associate to form a funcaccording to a standard nomenclature. The first three letters tional enzyme (alpha complementation). This type of complecorrespond to the genus and species of the host organism, folmentation can be recognized because these bacteria will form lowed by a strain identification or a letter indicating whether blue colonies when plated in the presence of a chromogenic the enzyme is encoded by an extrachromosomal bacteriophage substrate. However, if foreign DNA has been inserted at the or plasmid. and then by a Roman numeral if more than one plasmid polylinker site, the NH2-terminal 13-galactosidase fragrestriction endonuclease has been identified from the same host. ment is inactivated, there is no complementation. and bacteria carrying recombinant plasmids appear white.

axis yielding ends). Some

overlying enzymes

5 or cleave

3 single-stranded the two strands

protein. polylinker

Within this sequence

plasmid gene not interrupt

lies a conthe reading

Gene
To DNA ground cloning creasing

cloning Vectors
effectively technology, host and genomic todays of accomplish individual many genes of the must goals be of recombinant from steps of an backA variety of vector systems are now available to fulfill the in geneneeds of a particular experiment. Each has particular advantages ever-in- and disadvantages, some of which are listed in Table Bade-2. eachriophage vectors have played a particularly important role isolated

DNA. There are four basic investigator has the choice different methods for

number

accomplishing

in

RECOMBINANT-DNA

INTRODUCTION

251

Enhancer

Promoter

exon

exon

exon

Poly 3

A Site

5.

Transcription AAn AUG (Start)

NH2 UAG

RNA

Processing

(Stop)
COOH

Translation

(r U
FIG 2. Gene structure. A typical eukaryotic gene
mRNA transcript, and introns. or intervening sequences.

1
HO

Post
of exons.
are

Translational Modification

are sequences included in the mature from primary transcript during RNA processing. Promoter elements at the 5 end of the cluster focus RNA polymerase to the start site of transcription. Enhancer sequences increase transcription and characteristically function in either orientation. After traveling to the cytoplasm. the mRNA is translated into protein, with an adenine-uracil-guanine (AUG) codon usually serving as the
which spliced out the start site for this process.

is composed

which

Downloaded from www.ajcn.org by guest on May 31, 2011

molecular bacteria.
is a linear.

biology. The in assumes by host genome

double-stranded

Bacteriophages of the
DNA

are prototype
molecule

viruses

with

a tropism 50 of the are are

for These

cloning

vectors

can

be

classified

on

the

basis

of

whether

A-bacteriophage
approaching

vector the foreign (replacement kil12 bp (insertion of bacteria the size joined enzyme posfragment

DNA replaces a segment of nonessential vectors) or is directly inserted into the vectors). of the The fragment choice that the ofa needs particular to be vector cloned,

viral DNA viral genome is based the restriction the cloned bacteria. In cloning of which is necon

obases (kb) single-stranded the and sible. high with virus sealed

length. Each complementary a circular ligase. Two

terminus DNA. form when pathways

is composed Once inside the termini of replication

used to generate is to be expressed many next efficient

fragment. into protein vectors bacterial

and whether in the host allow promoter. the

During the (d/C the circular DNA is replicated copy number and the host cell eventually undergoes the release of infectious viral particles. Alternatively,

to

a this last regard lysis foreign inserts the essary to allow

expression to a strong transcription

E inco/i.

bacteriophage DNA may become integrated into the host bacterial DNA (lysogenic pathway). While in this state, the viral Gene DNA lies dormant, expressing only a few genes. Bacteriophages have been modified to accept and propagate exogenous DNA. The

isolation
isolation of genes can be isolated and approached as more it exists complex using in two basic

strategies. with its

The gene promoter and

can be elements

the genome intron-exon many a single

Axis 5
3

of symmetry -3

structure. introns

However, genes of higher eukaryotes are usually too large to be propagated

contain as

-JAATTC
-CTT:AAG
A

TABLE

-5

Cutting

DNA

molecules:

restriction

endonuclease

nomenclature

Enzyme5
EcoR tfho I I

Host I
Escherichia tlora.vel/a Nocardia

organism co/i
hosLs oiiiidis-caiaru,n

Target sequencet
GAATTC

RY 13

GATC
GC1GGCCGC

#{174}AATTC

____
FIG cleaves the DNA 3. Restriction double-stranded characteristically strands

Not

CTTAA
endonucleases. DNA display on either an at axis side well-defined of symmetry. of this axis. Each restriction sequences. and most endonuclease These enzymes sites

Sma
*

I The enzyme
isolated.

Serratia letters
Roman

inarcescens correspond
numerals

CCCGGG
to the initials
distinguish

of the host
among multiple

from

which
enzymes

it was

cleave isolated

from the same host. t G. guanine: A, adenine:

T. thymine:

and

C, cytosine.

252S
DNA

CARROLL
Endonuclease digestion of genomic DNA cDNA synthesis

fragments

I
Joining to vector
I

Ligation

of

LBIUnt end
_____________ ligation

Linker or adapter

cohesive termini

_____________ addition
Transfection of In vitro mRNA synthesis from cDNA. Injection into oocytes.

Introduction
into host cell

Transformation E. coil with recombinant plasmid

In vitro packang into phage coat. Transduction with [ge or cosmid

eukaryotic cells with expression vector

Selection recombinants

of

Nucleic
hybridization

acid

Immunochemical

PCR

Functional analysis, bioassays

exist to accommodate particular approaches

FIG

4. Steps

in

DNA

cloning.

An

ever-increasing

number

of alternatives

in the

four

basic

steps

in DNA

isolation

and

propagation. PCR.

polymerase

chain

reaction.

Adapted

from

reference

11.

Downloaded from www.ajcn.org by guest on May 31, 2011

unit. Antibiotic

Often

the

gene

is

cloned

laboriously

as

overlapping

frag-

ments. However, [complementary

it is possible DNA (eDNA)]

to

make ofthe

a double-stranded mRNA transcript

copy by use

RI digestion

ofthe enzyme reverse of introns. it contains untranslated ofcDNA Digested portions

transcriptase. only the limited

Because the protein-coding 5 and 6. or eDNA

mRNA is devoid sequence with The synthesis of the

to the

3 ends.

is illustrated in Figure total genomic DNA

representative

AATT TTAA

cells total mRNA creating a library When one wishes it is necessary to interest. Figure eDNA is cloned into infectious

can be inserted into whose contents contain to isolate a particular screen the library

a variety ofvector systems a host of different genes. gene from these libraries, to identify the species of

Anneay

7 illustrates a common strategy. In this example, in a bacteriophage vector that is then packaged viral particles. Each virus contains a different

TABLE Vectors

2 used

in DNA

cloning Library

DNA

Ligase

Size

of eDNA Genomic Comments

Recombinant Plasmid

0
I

Vector

insert kh

Recombinant
Plasmid

Plasmid
Transform Antibiotic
I

<10-15

Easy manipulation small fragments:

limited numbers

of

Bacteria selection

r cD c

):.( ..L
(,,>)

Bacterial Bacteriophage 1-23

of recombinants that can be screened.

Easy screening

chromosome

FIG 5. Generation of recombinant DNA molecules. Genomic DNA and Cosmid vector are cleaved with restriction endonuclease. The two species are incubated YAC together. allowing compatible termini to anneal to one another and become covalently linked by the enzyme DNA ligase. Bacteria are rendered competent to internalize the foreign DNA. and cells that gain the plasmid are selected by antibiotic resistance. RI. EcoR I restriction endonuclease.

35-50 50-400

of many recombinants. Screen like plasmid. Effective for genomic mapping, rapid chromosome
walking

RECOMBINANT-DNA

INTRODUCTION

253S

00 000
RNA AAAA--isolation AAAA DNA poly A + selection #{149} over oligo dT cellulose
5

cDNA Eco RI digestion

rI I r I

size selection

/\
----AAAA .-TTTT
I

ligase ATP

Linkers

3 R.T. dNTP T DNA

5 3

polymerase

______________

TTTT

I
.

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RNA

H/DNA

pOl 1

TTTT

FIG 6. eDNA synthesis. A DNA copy of the mRNA transcript can be synthesized by use of the enzyme reverse transcriptase. mRNA is isolated by binding of the poly-A tail to an oligo dT affinity column. An oligo dT primer is used to initiate first-strand eDNA synthesis in a 5 to 3 direction (anlisense) from the mRNA template. The RNA strand of the resulting heteroduplex is partially degraded by RNase H (RNA H). and DNA polymerase I initiates second-strand DNA synthesis (sense strand). with the final product being a double-stranded DNA copy ofthe mRNA. To ensure that the product has flush termini, the enzyme T7 DNA polymerase will polish uneven ends. This enzyme possesses both 5-to-3 polmerase activity and a 3-to-S exonuclease action that acts especially well on overhanging single-stranded DNA termini. The eDNA can then be modified to include any restriction sites at its termini by the addition of linker fragments. Linkers are short, blunt-ended. double-stranded fragments with convenient internal restriction sites. These are added to the DNA by DNA ligase (ie. blunt-ended ligation) and after digestion with the appropriate restriction endonuclease. yield cohesive termini capable of being ligated into the vector of choice. To ensure that this last digestion step does not cleave the eDNA itself if it happens to contain the same restriction site included in the linker, the eDNA may be methylated before the addition of linkers so that it is resistant to enzyme digestion. Newer versions of linkers called adapters obviate the need for these later steps. R.T., reverse transcriptase: dNTP. deoxynucleotide triphosphate.

phage host have lysis

orp/aque.

with bacteria

a unique and

gene plated on

insert. nutrient

These agar which

viruses media.

are

mixed Bacteria

withvectors that gene

contain transcription.

a strong In includes (/acZ). reading

bacterial most the systems promoter The frame

promoter a portion and

that a portion

directs ofthe operon /ac

foreignof the gene in the gene,

been infected or decreased on the onto

with growth lawn a piece

an individual ofthe host. of nitrocellulose

phage lead appears ofeach filter paper.

ofbacteria.

A sample

be replicated absorbs virus.

viral nucleic The filters are of DNA a piece of

acids as well as proteins screened with a radioactive to the desired itself, isolated

synthesized probe

segment may be different proteins. thetic small

homologous the gene

gene. Thus, the mRNA produced from this by the gene contains a short 13-galactosidase sequence at its 5 end, which that isallows a ribosome binding and efficient translation in the progene. The probe karyote host. The fusion protein produced by these recombinant previously from vectors a can be detected by radiolabeled antibody screening of

to eventual is used, which as a hole, for (.-galactosidase plaque can same translational The paper creating a fusiOti

eDNA insert is cloned as is the 3-galactosidase

cell type. a closely related gene from a family the same gene from a different host species. DNA fragment based on the protein sequence portion of the protein itself. The filter labeled probe will containing probe. Because bind, /irbridi:e, or After exposing the gene

of similar nitrocellulose filters in a fashion similar or a synnucleic acid probes. from a Newer methods ofscreening have been with vent some common described antibodies a fusion product problems above. directed protein, encountered Expression-vector against the or the similarly the bacterial to the way events isolation

to

that

described to circumthe traditional

for

developed with native host cloning protein may eukaryotic

is incubated

the radioactively mentarity, the ing and similar candidate plaques

sequences.

of sequence comple- techniques to plaques containbecause the filter to x-ray film, recognize can be identified the Such vectors One

is limited may not not modify cells do. for vec-

of interest

translated important method

isolated from the original master plate. Alternatively. eDNA molecules may be inserted into that allow efficient protein production E in co/i. These expression

posttranslational developed for molecules

include glycosylation. of cDNAs coding in Figure 8. Plasmid

cell-surface

is diagrammed

254S

CARROLL

cDNA
I

promoters 9). This vitro. inside

for permits Plasmid and the

RNA the the

polymerases production were the the current transcripts mRNA of clones resulting

flanking mRNA into were was for the presence divided

the from

cloning

(Figsite inserts was protein receptor The oocytes. mRNA

eDNA into into

ligate

to phage

arms
os

cos

in Once

pools, injected

produced,

oocyte

translated

I package

phage

and oocytes exhibited an eDNA divided After

containing electrical

mRNA in the

serotonin ofserotonin.

pool from which the transcript was produced was further until a single responsible clone was eventually isolated. isolating genes through these techniques, their nucleic

I Infect

host

E. coli

acid sequence can be determined and the complete identity of the translated protein can be deduced. Cloned genes can be used as probes themselves to analyze gene structure in different hosts or in certain such surveys ure 10. Genomic The which endonuclease. agarose gel. pathological states. The is called Southern blotting DNA is prepared DNA the and digested separates basic and digested technique is illustrated with used Fig- in in

a restriction on an to size electrode). many sites, transferred earlier, a

is then electrophoresed fragments according toward the positive genomic DNA at The DNA is then paper. As described labeled background through

phages on to nitrocellulose. Screen with radiactive probe

Lift

(smaller fragments migrate faster Because the enzyme cleaves total a broad smear of DNA is seen. from the gel to a piece of filter segment with the location If the sites, of the gene is radioactively filter. After washing away of the restriction more than DNA fragment endonuclease one band

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FIG 7. eDNA cloning. The eDNA with the addition of compatible ends is now ligated into the appropriate vector. In this example the vector is a bacteriophage. The phage is packaged into an infectious viral particle and is used to infect bacteria. Because the virus will eventually lead to
lysis single of the bacterial host. holes cell has will picked appear up a on the lawn of bacteria A where portion a of
,

EID
replicating within developed. been

and is incubated radioactivity, the autoradiography. at many if the

is seen

cleaves the gene internally may be detected. Alternatively,

SV4O on
splice
+

An

pBR

322

on

recombinant

virus.

these viral particles can be replicated onto a piece of filter paper can then be screened with a probe to identify the clone the DNA segment of interest.

paper. The containing

cDNA from antigen positive tissue

Eukaryotic transcription

unit

tors ganisms

capable have

of

eukaryotic These

cells vectors

of higher include or the Epsteinin eukaryotic

orviral

now

origins of replication. usually Barr virus, which allow their cells. These plasmids COS cells. ofmRNA can be such as expression

from the SV4O virus effective propagation introduced into sequences Translation

eukaryotic

Strong promoter from eDNA inserts.

induce proceeds,

hosts Transfect Into COS cells the

effective posttranslational cules are transported eDNA of interest can bodies recognizing the In some circumstances well-defined biological

modification takes place. and moleto the cell surface. Cells containing the Cells that express be enriched by stepwise binding to anti- antigen bind to Mab on plate cell-surface protein. a protein activity but may be associated may not have been with purified a

to allow the generation ofantibodies or limited peptide-sequence Cells harvested and plasmids analysis. One such example was the serotonin receptor. It was recovered for additional rounds of selection known that these receptors are expressed in the choroid plexus, FIG 8. Molecular cloning by cell-surface expression of protein. The which is a brain tissue associated with the production of cereeDNA is cloned into a vector that (eg, SV 40 ori virus) that is able to brospinal fluid. mRNA isolated from this tissue when injected replicate in eukaryotic cells. The eDNA is transcribed into mRNA and into frog oocytes was able to produce an inward electrical current translated into protein within the cell. The protein is modified and exin the presence of serotonin, an activity that occurs in vivo withported to the cell surface, whereit can be detected by a monoclonal serotonin binding. Researchers generated eDNA from the cho-antibody (Mab). An, poly A-addition site: pBR 322 ori, plasmid origin roid plexus and inserted it into a bacteriophage vector that has replication. of

RECOM positive Gradient Fractionation of RNA

.

BINANT-DNA

INTRODUCTION ization gene cleases are ofsueh and blotting of and the DNA. cleavage single-stranded many hairpin-loop conditions. the construction have amount It is even sources (eg. be in made of of newer. more-efficient less labor is spent abnormalities by portions called necessary character structures. Therefore. these Processed is not use different procedure and of ofthe Northern RNA is much before of RNA which the fine gene mapping of restriction as probes. blotting smaller eleetrophoresis. allows alter

255S ofthe normal endonuA variation analyzes than this strong vectorintensive, isolating a RNA native Howspecies its migration

accomplished

a series

_______________
Small RNA

ofthis
Large RNA

Fraction

Number

instead DNA ever, to form during

Construction from

of

Not!

cDNA expression

library
A T7 cDNA T3 A

positive fraction

electrophoresis.

gels run under are

denaturing Although

Divide cDNA clones intopools Transcription of cDNA pools in vitro with T7 RNA polymerase Injection of oocytes with cDNA transcripts and measurement of activity Purity cDNA by sequential dilution of cDNA
FIG 9.

I I I

cloning systems a considerable single from gene. different families may

these techniques laboratory effort to

more formidable (different species) genes). supply. many short

isolate the same or many members In addition. a technique

gene of has al-

CAP-RNA

gene material
Im
U

immunoglobulin greatly simplified (6). The polymerase amplification of DNA strands of

biological

Recently.

/u1OOnA

emerged that has DNA techniques lows

ingly

common recombinant chain reaction (PCR) from exceedoligomers flanking the

the efficient small amounts to opposite

of gene segments (FigI I). Single-stranded DNA at a point

Downloaded from www.ajcn.org by guest on May 31, 2011

bind

gene

pools
Functional analysis of eDNA clones. The eDNA

segment of interest. Substrate DNA ature to allow the strands to denature. lowered to allow the single-stranded
is inserted anneal

is heated to a high temperThe temperature is then oligonucleotide primers to temperature is high

to

the

target

DNA.

The

annealing

into a vector system where T3 and T7 RNA polymerase.

and lated ligand. is injected into Figure protein. adapted into oocytes. The

the cloning site is flanked mRNA is made in vitro

Within the containing oocyte the transcripts

by promoters from eDNA

transcripts for the mRNAs are

for pools
transreceptor

oocytes

are recognized containing

because

a current
from ref4.

is generated
CAP. terminus 5

in the I. current.

presence

of bound

5 3

Cycle
3.

ofeukaryotic

Denaturation, Primer DNA Synthesis Binding

7-methvlguanylate:

V. voltage:

gene is small a single hand by a deletion may produce

or ifonly a segment ofthe may emerge. A disruption or rearrangement a different pattern with other by this

gene is used as a probe, in gene structure caused chromosome technique. segments The local-

--

Amplified Segment

Cycle

Agerose
Gel

_
1234
-

::
-

Gel

5
.,-,--.,

*-

3
.

Acid (Depuririation) Base (Denaturatron)

Tn, (Neutralization)

1Treat-

of Gel merit

I
7
Hybridize

4
_______________

Paper Towels Filter Gel Sponge

xFilm

ExposetoFilm

__
Genomic a broad

FIG
restriction

10.

Southern endonuclease

blot

analysis. yielding

DNA smear of

is digested DNA on

with agarose

1 1 . Polymerase chain reaction. Single-stranded oligonucleotide bind to opposite strands of denatured DNA under stringent The gel is treated with an acid solution (depurinated) toprimers at high temperature. Taq polymerase functions at high temDNA and assist transfer of it to a section of filter paper. conditions to faithfully reproduce the gene segment of interest. Multiple The DNA is also denatured beforeit is transferred to allow binding of perature the target segment. Reproduced with permission from complementary radioactively labeled probe DNA. Reprinted with per- cycles amplify WL Carroll and AL Schwartz (12). mission from WL Carroll and AL Schwartz (2). electrophoresis. fragment the

a gel FIG

256S
Conventional Pulsed Field

CARROLL Repeated rounds oftarget to a number

review. The

of this DNA. of new

PCR

basic Several
reaction

reaction

lead too

to

logarithmic ofthis procedure

am-

(<20Kb) . . .

(4OKb-l400Kb) .

plification has
in

modifications
has had

led
this

applications DNA techniques.

numerous
a tremendous

to include
impact

on

routine

recombinant

The methods fragments up apparent that of kilobases. component

philia. approaches

discussed above are capable of analyzing DNA to a maximum size of 50 kb. However, it is now the genes ofhigher organisms may span hundreds For factor example, 8. which
200 kb.

the
The

gene
gene

encoding in
encoding

the patients

blood-clotting with
the I 5-kb

is deficient with

hemotran-

script trophy
#{149} + I + I + I +

that

is defective large gene band may In

in patients to cover > clusters In some lie a cannot

Duchennes kb). Effective may away.

muscular analysis using but

dysconvenits even-

is estimated techniques. location the

106 bp ( 1000 circumstances karyotype significant

ofthese tional tual

be approached a gene distance current analysis

be localized Walking

FIG I 2. Gel electrophoresis of DNA. In conventional electrophoresis the electric field is constant and oriented is in a parallel direction. Pulsedfield gel electrophoresis consists ofperpendicularly oriented. nonuniform. alternately pulsed electric fields.

to a particular toward practical. mapping humans. methods

in standard

gene of interest with recent years considerable

vector systems is iminterest has developed in organisms. including DNA fragments newer DNA can
enzymes

the genomes To effectively to isolate, map. oflarge

endonuclease

of many well-studied analyze larger and DNA

digestion

Downloaded from www.ajcn.org by guest on May 31, 2011

clone

large fragments
with

samples

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being

enough mentarity isolated aqualicus

to

allow only,
initiates

primer binding thus minimizing polymerase

new DNA copies

to sequences nonspecific from the

from the

of high binding. organismThermos

bound

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by primers. restriction

be accomplished
that recog-

heat-stable

nize

sequences

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appear

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infrequently

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arm Right arm

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FIG
for by arms. replication the

I 3.

Yeast sequence

artificial function: (ARSI). Two of

chromosome a centromere In addition. markers site cloning selectable the

(YAC) (CEN4). plasmid (TRPI (SUP4)

cloning

system. that that

The form

vector growth

consists in and of yeast with

of

three

yeast (TEL).

elements and an

essential autonomous both vector GF

chromosome thin line.

sequences sequences and leads to URA3) a red

telomeres

vivo

allow

selection E co/i arein represented colonies permission containing from DT Burke,

allow colony.

selection Reprinted

Interruption

Carle.

and

MV

Olson

(3).

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INTRODUCTION migrate varying through the time gel pores DNA effectively. have fields ofsueh propagation fragment 50 kb. is the functional segregation These 13). to yield isolated. in I:: co/i (Fig been with in (or the switch molecules Newer introduced fewer large in artifacts fragments vectors direction interval) of new between
>

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By

interval

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II
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in current direction. kb can be separated the the above field. The studies cause vectors such (3, 8). generation but their the largest approaches large fragments The that and sequences basic allow concept electric uniform

between 50 gel techniques and have due greatly remains to

featured distortion aids

of

Ai

mapping becosmid cloning (YAC) the with previously

a problem for

Ligation (dfute conditions)

accommodated One system yeast units and artificial of

by traditional now available chromosome yeast were capable appropriate linear DNA A recombinant Because are of a gene system DNA insertion

4,
I

chromosomes. were combined of replicating enzyme arms each plated yeast of

JJ
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Telomeres Yeast DNA pBR322 DNA Insert DNA Left Arm Sense Primer Left Arm Anti-Sense Primer

sequences identified plasmid digestion. to accept

Genomic End Rescue PCR

replication. vector the two DNA.

elements After

a hybrid

as a plasmid

the circular vector yields a large segment offoreign used to transform yeast a selectable marker, media, both between leads to which vector both distinctive allows arms. In vector red propagate of comprehensive to the isolation of reaction

able clone arm on eon-

4,

can be contains defined taming fragment which been to allow

spheroplasts. the transformants selective addition. outgrowth the

Downloaded from www.ajcn.org by guest on May 31, 2011

a DNA

arms interrupts colonies. This enough large large libraries. DNA illustrated

sequence, has now fragments strategy eon14

shown already

to faithfully construction led

This fragments in

! 4
PCR Product
*

has taming An

multiple application

gene clusters. of the PCR

Figure

kg.m

ao

r.oa.

allows the rapid isolation into YACs. These ends contiguous a jump

ofthe ends ofthese can then be used library. and of interest frames genes can This type

large inserts cloned as probes to isolate to that protein of genetic re-

FIG I 4. Inverted PCR to isolate ends of YAC clones. The YAC clone is digested with enzyme TaqI. which cleaves the left vector arm at known site adjacent to the cloning site and the insert at an unknown site within the clone. The TaqI fragments are ligated together to form circle. This lines up two primers into position to be able to amplify insert DNA by the PCR reaction. Reprinted with permission from GA Silverman. RD Ye, KM Pollock. JE Sadler. and Si Korsmeyer (9).

clones from the YAC to one end ofa fragment toward for not greatly diseases. been aid a gene new identified. our open-reading

Thus, one can efficiently use the ends as probes (9). and By scanning abnormalities whose reverse or ofanalysis, ofa variety these

rapidly walk a large fragments occur products

genetics,

in certain have will

be isolated

understanding

combinant DNA technology and introduces some more recent this size has a tendency to shear when standard digestions in methodological advances to allow more efficient, reliable gene solution are used, it is necessary to carry these reactions out in cloning. The power ofthese techniques makes it mandatory for agarose. These agarose blocks are then loaded directly into slots every life scientist to be familiar with this field. This knowledge at the origin of horizontal agarose gels. Newer electrophoresis will not necessarily provide investigators with right the questechniques allow separation of such large DNA molecules. In tions but will likely solve biological problems not approached conventional gel eleetrophoresis the separation ofDNA is based by previous techniques. The thoughtful application of this on the sieving properties of the gel matrix. A smaller molecule knowledge in the field of biotechnology will undoubtedly lead easily negotiates the pores of the gel whereas larger molecules to solutions to the problem ofan increasing food demand from have greater difficulty. However, under the influence ofan eleea planet whose resources are already strained. #{163}3 tive field, the DNA molecule may change its shape by assuming an elongated referred gels, configuration parallel In conventional to the electric a certain oftheir the field, a process size of the to as replalion. continuous-electric-field threshold length. Pulseddirection

DNA with

fragments enzymes

of such

100 to 1000 kb may be seen after > as%1/u I, Not I, and X/zo I. Because

digestion DNA of

diseases This

and will allow article provides

more efficient mapping a simple overview

oflarge genomes. of conventional

(>
field

reptating DNA molecules above 30 kb) all migrate together independent ge/ e/ectrophoresis involves alternating current (Fig 12). to this change time

References

electric response take

DNA molecules reorient themselves in current direction. Larger molecules themselves before they effectively

a longer

to reorient

1 . Aruffo A. Seed B. Molecular cloning of a CD28 eDNA by a highefficiency cos cell expression system. Proc Natl Acad USA Sci in 1987:84:8573-7. 2. Berger SL, Kimmel AR. eds. Guide to molecular cloning techniques. Methods Enzymol. San Diego. CA: Academic Press, l987.

258S

CARROLL Burke ogenous Science DT. Carle GF. Olson M. Cloning DNA into yeast by means ofartificial 1987:236:806-12. DC. Cantor CR. Separation of yeast of large segments of ex- 8. Schwartz DNAs by pulsed field gradient gel eleetrophoresis. chromosome vectors.
75.

3.

chromosome sized Cell 1984:37:67-

4.

Julius D. MacDermott terization of a functional Science 1988:241:558-64. Lewin B. Genes

AB. Axe! R. Jesse!! TM. Molecular characeDNA encoding the serotonin Ic receptor. York: Oxford University Press. 1990.

5.

IV. New

6.

Saiki RK. Gelfand DN. Stoffel S. et al. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988:239:487-9!. I. Fritsch EF. Maniatis T. Molecular cloning. manual. Cold Spring Harbor. NY: Cold Spring Harbor Press. 1989. A laboratory Laboratory

7. Sambrook

GA. Ye RD. Pollock KM. Sadler JE, Korsmeyer Si. Use artificial chromosome clones for mapping and walking within human chromosome segment 18q2 I .3. Proc NatI Acad Sei USA 1989:86:7485-9. 10. Darnell J, Lodish H. Baltimore D. Molecular cell biology. New York: Freeman WH, 1986. 1. Old RW, Primrose SB. Principles of gene manipulation. Cambridge, UK: Blaekwell Scientific Publications. 1985. 12. Carroll WL. Schwartz AL. Molecular oncology. In: Fernbach Di, Vietti Ti. eds. Clinical pediatric oncology. St Louis: CV Mosby. 199 1:29-68.
ofyeast

9.

Silverman

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