Download Amplification of DNA Sequences

CE U P D A T E — M O L E C U L A R
BIOLOGY
III
James Wisecarver, MD, PhD
Amplification of DNA Sequences
The techniques for studying intact, high-molecular-weight DNA obtained from tissues, discussed
earlier in this series, require DNA with ample
numbers of target sequences. In some instances,
however, only a small amount of material might
be available for study (eg, a few cells from a fineneedle aspirate) or only a few target sequences
available for detection (eg, a few viral particles in
a sample of cerebrospinal fluid). In these cases, it
is useful to selectively amplify the DNA target
sequence of interest, thereby providing ample
copies of the DNA sequence for study. This article will discuss methods for producing multiple
copies of DNA sequences and techniques for
detecting variations in nucleotide sequences,
including DNA sequence analysis.
ABSTRACT \This article discusses some of the tools
available to amplify DNA sequences. These techniques are
extremely useful for detecting the presence of small amounts
of a DNA target sequence. They also can he used to detect
short sequences in DNA that has degraded partially. The
cloning procedure, useful for producing many copies of a
sequence, also is described. Methods for comparing
nucleotide sequences within amplified sequences are
discussed, including heteroduplex analysis and single-strand
conformational polymorphism (SSCP) analysis. Methods for
performing DNA sequence analysis are presented as well.
This is the final article in a three-part series on DNA. Other articles discussed the
structural properties of DNA, how it is extracted from cells for study, some of the
basic tools used to gain useful clinical information, and the techniques commonly
DMA Amplification Techniques
used to test native high-molecular-weight DNA obtained from cells and tissues. On
o
The probe hybridization technology mentioned
earlier requires high-molecular-weight DNA
from a large number of cells. Each cell contains
only one copy of the DNA. Oligonucleotide
probes require that many copies of the target
DNA be present to provide enough hybridization
points to permit target localization on a blot. In
similar fashion, in-situ hybridization works best
when multiple viral or other target sequences are
present within each cell, again providing a sufficient number of sites for probe hybridization to
permit detection of the signal. In other instances,
however, only a few copies of the target sequence
may be present. In such cases, it is desirable to
amplify this limited amount of target sequence so
that it can be detected and studied.
Strategies recently have been developed to
amplify a single target sequence millions of times
so that it can be detected more easily. The most
widely used amplification strategy is the polymerase chain reaction (PCR). To use this procedure, the target sequence to be amplified must be
completion of this series, readers will be able to describe the composition of DNA,
c
how the strands are arranged, how to extract DNA from tissues prior to testing, and
how DNA fragments are prepared by enzyme digestion, separated using gel elec-
u
known. Typically, this target sequence ranges
from 100 to 1,000 base pairs in length. To perform PCR, two small oligonucleotide primer
sequences must be synthesized that typically are
16 to 20 base pairs in length. These primer
sequences are complementary to the 3' ends of
the sequence to be amplified (Fig 1).
Initially, the DNA is denatured by heating in
the presence of the synthesized primer sequences.
As the reaction mixture cools, some of the DNA
strands reanneal with their original complementary partner strands. Because the synthetic primer
pieces are present in excess numbers, however,
they are more likely to bind to the complementary
region on the native DNA strand. With the primers
bound to the native DNA, the polymerization
(DNA synthesis) reaction is free to proceed. The
technologic breakthrough that permitted the
automation of this technique was the discovery of a
VOLUME 28, NUMBER 3
E
E
0
trophoresis, and then isolated for further study.
MARCH 1997
3
From the
Department of
Pathology and
Microbiology,
University of
Nebraska Medical
Center, Omaha.
6
I
Reprint requests
to Dr Wisecarver,
Department of
Pathology and
Microbiology,
University of
Nebraska Medical
Center, 600 S 42nd
St, Omaha, NE
68198-3135.
LABORATORY MEDICINE
191
The Polymerase Chain Reaction
DNA sequence to
be amplified
1
Denaturation
Primers bind to
complementary regions.
1
N e w DNA strands are synthesized.
3'
y Newly synthesized D N A \
3'
5'
1
5'
3'
Now two copies of
sequence serve
as template.
\
5'
3'
5'
Primer binding
5J
5'
1
3'
5;
3'
5'
3'
3'
5'
\
3'
5'
DNA synthesis
5'
5'
3'
3'
5'
5'
After two cycles, four copies of original sequence are available to serve as templates
for synthesis of additional strands during the next cycle.
Fig 1. Polymerase
chain reaction.
After determining a
sequence of interest,
short, 15-to-20
base-pair primer
sequences are synthesized that are
complementary to
the 3' end of each
strand. After the
native, doublestranded DNA has
been denatured, the
primer strands bind
to the denatured
strands and serve as
a start point for DNA
synthesis. The reaction is catalyzed by a
thermostable DNA
polymerase enzyme
that is resistant to
the high temperatures needed to
denature the newly
synthesized strands
so that the second
cycle of the reaction
can occur.
192
DNA polymerase enzyme from a strain of bacteria
collected from a mineral hot spring. These bacteria
are capable of surviving at high temperatures, and
their biologic enzymes are capable of withstanding
temperature extremes. Consequently, this enzyme
does not lose its ability to synthesize new DNA
despite numerous denaturation steps. After the
primer sequences have attached, or annealed, to
the target DNA strand, the DNA polymerase
enzyme will generate new DNA strands by
incorporating individual nucleotide bases that
are provided in the reaction mixture (Fig 1).
After synthesis of the new DNA strands, the
reaction mixture again is heated to 96°C to
denature the newly generated DNA pieces from
the original DNA strands. These new DNA
strands, along with the original strands, can
serve as templates for additional amplification
cycles. If these steps are repeated 25 to 30 times,
it is possible to amplify the original DNA segment 1 million times. Millions of copies of the
sequence can be generated within a few hours.
As outlined above, this amplification process
provides large numbers of copies of the original
target sequence. Often, it is necessary only to
LABORATORY MEDICINE
VOLUME 28, NUMBER 3
MARCH 1997
determine whether amplification occurred. For
example, you can detect viral DNA from a blood
sample using a pair of primers specific for a short
sequence within the viral genome that is specific
to the virus and is not found in mammalian cells.
Following amplification, the presence of viral
DNA would yield a PCR product of the appropriate size; whole samples not containing viral DNA
would have no product. These amplified products
can be detected simply by placing the PCR reaction mixture in agarose gel and performing electrophoresis. Because the length of the DNA target
sequence is known, the presence of a distinct band
in the agarose gel following ethidium bromide
staining that is of the appropriate size is evidence
that viral DNA was present in the sample.
To further confirm that this is viral DNA, the
amplified fragments can be transferred to a
membrane using the Southern blot procedure,
and an oligonucleotide probe, specific for a
sequence within the viral PCR product, is applied
to confirm that the amplified product is virus
DNA rather than nonspecific amplification of
human DNA.
In the case described, the presence of a band is
evidence that the viral sequence was present in
the specimen. The absence of a band is problematic, however, in that one cannot be certain
whether the sequence indeed was absent, or that
the reaction conditions were inappropriate for
the PCR reaction to proceed. In most cases, a
positive control reaction is used as well, in which
a second set of primers to a normal human gene
certain to be present are added to ensure that the
PCR conditions were adequate and that all
reagents were added. Techniques based on the
PCR are being applied to clinical situations to
detect both human and microbial genes within
tissues and blood specimens from patients.
These amplification techniques can be performed relatively quickly and require only a small
amount of DNA. The Southern blot procedure
requires DNA from many cells and up to several
weeks to perform. The PCR technique, using
rapid and simplified DNA isolation procedures,
can be performed in only a few hours using DNA
from a small number of cells. Potentially, results
can be available the same day the patient's sample
is received in the laboratory. Also, the increased
sensitivity of amplification procedures allows for
the detection of a very small number of copies of
a target sequence in a relatively large specimen
(eg, a few copies of a viral sequence in a sample
of cerebrospinal fluid).
DNA Cloning
Gene Cloning
One of the more important advances in molecular biology has been the ability to remove segments of mammalian DNA and insert them into
small circular portions of bacterial plasmid DNA.
This technique involves removing the DNA
sequence of interest by cutting it away from the
intact genomic DNA using restriction enzymes.
This DNA restriction fragment is inserted into a
circular piece of plasmid DNA for insertion into
bacteria (Fig 2).
The bacteria then are propagated in culture
using a system that is selective for the bacterial
organisms containing the plasmid DNA. This
often is achieved using a plasmid vector containing an antibiotic resistance gene. The bacteria
then are cultured in the presence of the antibiotic.
Only bacteria containing the plasmid with the
gene to inactivate the antibiotic are capable of
sustained growth within the medium. Using this
approach, large numbers of copies of the DNA
target fragment can be generated as the bacteria
multiply. The human DNA fragments can be isolated by lysing the bacteria using a special buffer,
collecting the plasmid DNA, and then excising
the human portion using the original restriction
enzyme. The DNA sequence of the human fragment can be determined as outlined below. Using
bacterial plasmids, only small portions of human
DNA can be inserted. For larger sequences, other
cloning carriers (vectors) have been developed
(eg, yeast artificial chromosomes).
D N A Sequencing
Sometimes, it is desirable to know the entire
nucleotide sequence of a particular portion of
DNA. This is especially useful when looking for
point mutations (single nucleotide changes)
within a gene encoding a biologically important
protein. Sequence analysis requires a large number of copies of the particular sequence of interest. These copies can be obtained either by
cloning the sequence into a plasmid vector,
inserting the vector into competent bacteria and
amplifying it, or by using PCR to produce large
numbers of copies of the target sequence. After
the sequence has been amplified, a primer is synthesized that will hybridize at the 3' end of the
DNA strand to be sequenced. Individual
nucleotides now are added along with a DNA
polymerase enzyme. In addition, one of the four
bases (ie, adenine, thymidine, cytosine, or guanine) is labeled with a radioactive marker. A
small amount of a single nucleotide is added as a
Human DNA (Double stranded)
Circular bacterial
plasmid DNA
(Double stranded)
Treat with restriction enzyme
Plasmid ring is opened.
DNA containing region of
interest is cut out.
Human DNA
Circular plasmid DNA containing human insert
di-deoxyribose derivative. Four separate reactions
are set up, each containing a different labeled
base and di-deoxyribose terminator. The advantage of using these di-deoxynucleotides is that
there is no hydroxyl group at the 3' position to
permit chain extension. Wherever one of these
labeled bases is incorporated, the growing
nucleotide strand is terminated. By providing an
excess of unlabeled single deoxy bases, the
chances of incorporating the di-deoxy chain terminators is a random event. A large number of
molecules will incorporate the regular deoxy
nucleotides and the strand extension will continue.
Occasionally the di-deoxy terminator will be
incorporated, resulting in chain termination. The
four reaction mixtures then are separated electrophoretically in an acrylamide gel with each
lane representing a different labeled base. Using
the appropriate acrylamide concentration, it is
possible to separate the individual nucleotide
fragments on the basis of size, each being one
base longer than the previous fragment. Using
this method, it is possible to read off the sequence
directly from the gel.
Automated methods have been developed that
incorporate fluorescent labels into the di-deoxy
MARCH 1997
VOLUME 28, NUMBER 3
Fig 2. DNA cloning
strategy. A fragment
of interest is isolated
from human DNA
using restriction
enzymes. The
excised fragment is
inserted into the circular plasmid DNA
vector that has been
cut open using the
same restriction
enzyme used to prepare the human
fragment. The plasmid DNA containing
the human insert is
then incorporated
into bacteria, usually
Escherichia coli,
where it is copied as
the bacteria replicate. Using this
approach, large
numbers of copies
of the human DNA
insert can be
obtained.
LABORATORY MEDICINE
193
Fig 3. Single-strand
conformational
polymorphism.
Following denaturation, as the nucleic
acid strands are
allowed to cool, they
assume a threedimensional structure based on internal areas of complementary sequence,
and consequently
fold up into hairpinlike structures. These
differences in threedimensional conformation affect the
migration of the
DNA fragments in
acrylamide gel. Two
polymerase chain
reaction products
that have an identical migration pattern
are assumed to be
identical, while products that migrate differently have areas
where the sequences
differ.
Single-Strand Conformational Polymorphism
Product A
I
Polymerase chain
reaction products:
double-stranded DNA
Product B
Heat to denature.
As mixture cools, the individual
strands fold to form hairpin-like
structures based on internal areas
of base complementarity.
I
t e r m i n a t o r s . Using this
approach,
each
chain
t e r m i n a t o r can be labeled
with a different color and all
of the reactions can be run
in a single lane on a gel. A
laser scans the gel, determining which color terminator is
p r e s e n t at each l o c a t i o n .
Using this method, the software within the instrument
is able to d e t e r m i n e the
sequence with a high degree
of accuracy.
Single-Strand
Conformational
Polymorphism
Single-strand conformational
p o l y m o r p h i s m (SSCP) is
Products migrate differently based
used to detect subtle variaon alterations in three-dimensional tions in short DNA sequences
structure.
such as PCR-amplified products. This technique typically
is used to compare sequences
from two or m o r e individuals to d e t e r m i n e
whether they are identical or whether a mutation
has occurred. To perform this type of analysis, the
amplified products are denatured into single
strands and then separated using acrylamide gel
The following guide will help you in your study of DNA amplification
electrophoresis. Following denaturation, as the septechniques.
arate strands cool, they fold up on themselves,
assuming a three-dimensional conformation based
Amplification—A process to produce multiple copies of a specific DNA
on intrastrand regions of base complementarity. If
sequence.
there are focal base differences within the same
Di-deoxyribose derivative—A ribose sugar in which hydrogen replaces the
sequence s e g m e n t from two individuals, or
hydroxyl groups at both the 2' and 3' positions on the ring.
between two different allelic forms of gene from
Gene cloning- -A method for producing quantities of a specific DNA
the same individual, these strands fold up in
sequence.
slightly different conformation due to these complementarity
differences. These conformational
Heteroduplex analysis—A modification of single-strand conformational
differences
can
be seen when the strands are seppolymorphism that can be used to determine sequence differences in allelic
arated using acrylamide gel electrophoresis (Fig
variants of a gene.
3). Single-strand conformational polymorphism
Nucleotide sequencing—A method to determine the entire nucleotide
has
been used to detect m u t a t i o n s in normal
sequence of a particular portion of DNA. Sequence analysis requires a large
human
genes such as the p53 tumor suppressor
number of copies of the particular sequence of interest.
gene.
Plasmid vector—A piece of circular DNA contained within bacterial organA modification of SSCP can be used to deterisms. Under appropriate conditions, this plasmid can be introduced into bacmine sequence differences in allelic variants of a
terial organisms, bringing with it additional genetic information.
gene. This technique is called heteroduplex analyPolymerase chain reaction (PCR)—A strategy that uses synthesized oligonusis and involves amplifying the genetic locus of
cleotide primers to amplify a single target DNA sequence so that it can be
interest using PCR. The amplified products are
detected and studied more easily.
m i x e d t o g e t h e r , d e n a t u r e d by h e a t i n g , a n d
allowed to cool. In some cases, the original two
Single-strand conformational polymorphism—A technique used to detect
strands from each amplicon reanneal to their
subtle differences in nucleotide sequences; typically used to compare
sequences from two or more individuals to determine whether they are idencomplementary partner (homoduplex). In other
tical or whether a mutation has occurred.
instances, the strand from one allele binds to a
A Guide to Amplification Terminology
194
LABORATORY MEDICINE
VOLUME 28. NUMBER 3
MARCH 1997
strand from the other nonidentical allele (heteroduplex). While there is a high degree of
sequence complementarity, the match is not perfect, leading to a short region or regions that are
not bound, thereby altering the electrophoretic
migration. By analyzing the reaction mixture
using an acrylamide gel, it is possible to separate
these heteroduplexes from the homoduplexes,
indicating that some areas of noncomplementary
sequence are present and therefore that these
pieces of sequence are not identical.
Potential Uses
The techniques outlined in this series have a wide
range of clinical uses within the laboratory. The
most obvious and exciting applications are in the
area of clinical microbiology. Using sensitive
amplification-based strategies, small amounts of
microbial DNA can be detected in clinical samples.
These techniques also have wide applicability to
hematopathology. The Southern blot techniques
and, more recently, PCR-based assays have been
used to detect clonal rearrangement of
immunoglobulin and T-cell receptor genes, indicating the presence of a clonal proliferation of
lymphocytes. Detection of a clonal expansion of
lymphoid cells suggests the presence of a lymphoid
neoplasm. Other PCR techniques have been
developed to detect chromosomal translocations
typical of other lymphoid and hematopoietic
malignancies, as well as some types of solid
organ neoplasms.
Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols:
A Guide to Methods and Applications. San Diego, Calif:
Academic Press; 1990.
Piper MA, Unger ER. Nucleic Acid Probes: A Primer for
Pathologists. Chicago, 111: ASCP Press; 1989.
Ross DW. Polymerase chain reaction. Arch Pathol Lab Med.
1990; 114:627.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A
Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press; 1989.
Please let us know your opinion of t h e
Molecular Biology (702) series.
1. T h e series m e t t h e objectives stated in t h e
abstract.
Deficient
1
Excellent
5
Test Time
Look for the CE
Update exam on
Molecular Biology
(702) following this
article. Participants
will earn 3 CMLE
credit hours.
2. T h e series p r o v i d e d useful technical data or
o r i g i n a l ideas.
Deficient
1
Excellent
5
3. T h e i n f o r m a t i o n p r o v i d e d in t h e series w a s
new and timely.
Deficient
1
Excellent
5
4. Technical p o i n t s w e r e explained clearly a n d
w e r e easy t o c o m p r e h e n d .
Deficient
1
Excellent
5
5. T h e text w a s organized logically.
Deficient
1
2
3
4
Excellent
5
6. Illustrations, charts, a n d tables helped
explain text a n d a d d e d t o series value.
Deficient
1
2
3
4
Excellent
5
C o m m e n t s : (attach a d d i t i o n a l pages, if
necessary)
0
e
3
Summary
An increasing number of molecular tests are
being developed, and the results are beginning to
appear in the literature monthly. Virtually all
tests are based on exploiting the physical and
chemical properties of DNA using techniques
such as DNA amplification, gene cloning, and
nucleotide sequencing. An understanding of the
basic concepts of nucleic acid structure and physical properties will help the reader evaluate these
reports for possible clinical significance.
Eventually, these techniques will find their way
into most clinical laboratories. A basic understanding of these concepts will aid the pathologist
or technologist in analyzing the results and troubleshooting the test procedures.©
Selected Readings and Reference Materials
E
E
o
0
T h a n k y o u f o r y o u r input. M a i l this f o r m w i t h
y o u r e x a m or alone t o :
Laboratory
Medicine, 2100 W Harrison St,
Chicago, IL 60612-3798.
c
.2
'o
©
Correction
In the first article of this series, titled "The ABCs of DNA" {Lab Med.
1997;28:48-52), the CH groups in Fig 1 on page 49 should be hydroxyl
(OH) groups. The corrected figure follows.
5"
OH —CH 2
Base
V
4"
OH
3"
OH
Y
5"
OH—CH 2
Base
r
4"
OH
3"
H
2'
Deoxyribose
Brown TA. DNA sequencing: The basics. Oxford, England:
Ribose
Oxford University Press; 1994.
Glick BR, Pasternack JJ. Molecular Biotechnology: Principles
and Applications of Recombinant DNA. Washington, DC: ASM Fig 1. Structural comparison of ribose and deoxyribose. The only difference in
Press; 1994.
structure is the exchange of a hydrogen molecule for the hydroxyl group
attached to the carbon molecule in the 2' position.
MARCH 1997 VOLUME 28, NUMBER 3
LABORATORY MEDICINE
195
E
0