ASTM E1873-97(2002)
(Guide)Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique
Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique
SCOPE
1.1 This guide covers guidelines, recommendations, basic considerations, criteria, and principles to be employed when developing, utilizing, or assessing PCR procedures and specific protocols for the amplification and detection of nucleic acid sequences. This guide is not intended to be a standard procedure with a list of requirements for PCR detection of nucleic acids. This guide is intended to provide information that will assist the user in obtaining quality and reliable data.
1.2 Nucleic acid sequences that can be amplified by PCR include RNA, as well as DNA sequences; RNA sequences are suitable targets for PCR following reverse transcription of the RNA to complementary DNA (cDNA). This type of amplification technique is often called reverse transcription-PCR (RT-PCR).
1.3 This guide has been developed for use in any molecular biology/biotechnology laboratory. This includes, but is not limited to, laboratories that specialize in the diagnosis of human, animal, plant, or bacterial diseases.
1.4 This guide conveys the general procedural terminology of PCR technology used for the detection of nucleic acids.
1.5 This guide is a general one; it does not cover the additional guidance that would be needed for specific applications, for example, for the PCR detection of nucleic acid sequences of specific microorganisms.
1.6 This guide does not cover details of the various methods that can be utilized to identify PCR-amplified DNA sequences.
1.7 This guide does not cover specific variations of the basic PCR or RT-PCR technology (for example, quantitative PCR, multiplex PCR, and in situ PCR), and it does not cover details of instrument calibration.
1.8 Warning—Laboratory work involving certain clinical specimens and microorganisms can be hazardous to personnel. Precaution—Biosafety level 2 facilities are recommended for potentially hazardous work (8). Safety guidelines should be adhered to in accordance with NCCLS M29-T2, I17-P, and other recommendations (8).
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Designation: E 1873 – 97 (Reapproved 2002)
Standard Guide for
Detection of Nucleic Acid Sequences by the Polymerase
Chain Reaction Technique
This standard is issued under the fixed designation E 1873; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide applies to the detection of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
sequences by the polymerase chain reaction (PCR) technique. The PCR is used as a tool in many
molecular biology laboratory settings and for diverse reasons, for example, for amplification and
detection of nucleic acid sequences and for DNA sequencing. There is an abundance of publications
addressing laboratory procedures and specific protocols for various applications. The field of PCR is
advancing so rapidly; however, it is necessary to frequently modify and update these procedures and
specific protocols. Rather than describe a standard procedure and list requirements that would
probably soon become outdated, this guide consists of guidelines, recommendations, basic consider-
ations, criteria, and principles that should be employed when developing, utilizing, or assessing PCR
procedures and specific protocols for the amplification and detection of nucleic acid sequences.
This guide was developed by Subcommittee E48.02 on Characterization and Identification of
Biological Systems in collaboration with DIN (German Institute for Standardization) Committee E9
on Serodiagnosis of Infectious Diseases and Diseases of the Immune System, Department for Medical
Standards (NAMed).
This guide assumes a basic knowledge of microbiology and molecular biology. It assumes the
availability of basic references in PCR for general procedures (see Refs 1-7) and the ability to search
the literature for target-specific protocols.
1. Scope RNA to complementary DNA (cDNA). This type of amplifi-
cation technique is often called reverse transcription-PCR
1.1 This guide covers guidelines, recommendations, basic
(RT-PCR).
considerations, criteria, and principles to be employed when
1.3 This guide has been developed for use in any molecular
developing, utilizing, or assessing PCR procedures and specific
biology/biotechnology laboratory. This includes, but is not
protocols for the amplification and detection of nucleic acid
limited to, laboratories that specialize in the diagnosis of
sequences. This guide is not intended to be a standard
human, animal, plant, or bacterial diseases.
procedure with a list of requirements for PCR detection of
1.4 This guide conveys the general procedural terminology
nucleic acids. This guide is intended to provide information
of PCR technology used for the detection of nucleic acids.
that will assist the user in obtaining quality and reliable data.
1.5 This guide is a general one; it does not cover the
1.2 Nucleic acid sequences that can be amplified by PCR
additional guidance that would be needed for specific applica-
include RNA, as well as DNA sequences; RNA sequences are
tions, for example, for the PCR detection of nucleic acid
suitable targets for PCR following reverse transcription of the
sequences of specific microorganisms.
1.6 This guide does not cover details of the various methods
that can be utilized to identify PCR-amplified DNA sequences.
This guide is under the jurisdiction of ASTM Committee E-48 on Biotechnol-
ogy and is the direct responsibility of Subcommittee E48.02 on Characterization and
1.7 This guide does not cover specific variations of the basic
Identification of Biological Systems.
PCR or RT-PCR technology (for example, quantitative PCR,
Current edition approved April 10, 1997. Published June 1997.
multiplex PCR, and in situ PCR), and it does not cover details
The boldface numbers in parentheses refer to the list of references at the end of
this standard. of instrument calibration.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E 1873 – 97 (2002)
1.8 Warning—Laboratory work involving certain clinical 3.2.7 extension (of primer), n—in PCR, the synthesis of a
specimens and microorganisms can be hazardous to personnel. new strand of DNA that is complementary to the target DNA,
Precaution—Biosafety level 2 facilities are recommended for
used as a template, by the addition of nucleotide triphosphates
potentially hazardous work (8). Safety guidelines should be
to the oligonucleotide primer annealed to the target DNA.
adhered to in accordance with NCCLS M29-T2, I17-P, and
Extension is catalyzed by an enzyme with DNA polymerase
other recommendations (8).
activity.
3.2.8 hot-start PCR, n—a variation of PCR designed to
2. Referenced Documents
minimize the formation of non-specific amplification products,
2.1 NCCLS Standards:
often exhibited by smearing on gels, that may occur during the
I17-P Protection of Laboratory Workers form Instrument
reaction setup, thereby enhancing the specificity, sensitivity,
Biohazards; Proposed Guideline (1991)
and precision of the amplification reaction. There are several
C24-A Internal Quality Control Testing: Principles and
3 ways to achieve hot-start PCR. All methods involve withhold-
Definitions; Approved Guideline (1991)
++
ing a critical component (for example, polymerase or Mg )
M29-T2 Protection of Laboratory Workers from Infectious
during the reaction setup at room temperature. Reaction tubes
Disease Transmitted by Blood, Body Fluids, and Tissue—
are then heated to a temperature exceeding 60°C, at which
Second Edition; Tentative Guideline (1991)
point the critical component is either added to the tube, comes
GP5-A Clinical Laboratory Waste Management; Approved
in contact with the reaction mixture (for example, a hot start
Guideline (1993)
MM3-A Molecular Diagnostic Methods for Infectious Dis-
wax), or is activated (for example, a DNA polymerase used for
eases; Approved Guideline (1995)
hot start PCR).
3.2.9 hybridization, n—the specific annealing of a comple-
3. Terminology
mentary DNA strand (for example, an oligonucleotide DNA
3.1 Basic concepts for quality assurance in the laboratory
primer) to the target DNA or RNA or amplified DNA segment
should be followed, in accordance with NCCLS C24-A.
in a medium containing an appropriate buffer composition, pH
3.2 Definitions of Terms Specific to This Standard:
value, and temperature range.
3.2.1 annealing, v—in PCR, the binding or hybridization of
3.2.10 nested PCR, n—a modification of PCR that uses
two complementary strands of DNA or of DNA and RNA, as
nested sets of PCR primers to enhance the sensitivity and
in the hybridization of oligonucleotide primers with DNA or
specificity of the reaction. A nested PCR protocol consists of
RNA.
two rounds of PCR amplification and utilizes two sets of
3.2.2 carryover contamination, n—in PCR, the adulteration
primer pairs. In the first round of amplification, the outermost
of a PCR with amplified material (PCR products) from a
primer pair is used to generate an amplification product that is
previously amplified sample or with nucleic acids from another
then subjected to a second round of amplification in the
source. This potentially can cause a false positive result.
3.2.3 chaotrope, n—in PCR, a chemical denaturing agent presence of the internal primer pair.
that lowers the melting temperature of double-stranded nucleic
3.2.11 polymerase chain reaction, PCR, n—an in vitro
acids.
laboratory method for the enzymatic amplification of nucleic
3.2.4 denaturation, n—in PCR, separation of double-
acid sequences (1-3). Two DNA oligonucleotide primers an-
stranded DNA into single-stranded DNA. This can be accom-
neal with their complementary DNA strands and flank (that is,
plished with heat, extremes of pH, or exposure to certain
border) the segment to be amplified. The increase in amount
chemicals such as chaotropic agents, plus heat, usually.
(amplification) of the DNA segments occurs during repeated
3.2.5 DNA polymerase, n—for PCR, usually a thermostable
cycles consisting of three steps: heat denaturation of the
enzyme used in PCR that catalyzes the repeated synthesis of
double-stranded DNA, cooling to effect annealing of the
DNA under the selected reaction conditions of the method. The
primers to their complementary DNA strands, and enzymatic
PCR, however, also can be performed with DNA polymerases
extension of the annealed primers by DNA polymerase at an
that are not thermostable, if fresh enzyme is added before each
optimal temperature. The amplification results in a near expo-
cycle.
nential increase in the amount of the nucleic acid target defined
3.2.6 DNA probe, n—for PCR or RT-PCR, an oligonucle-
by the primers.
otide that is complementary to a portion of the amplified DNA
product, contains a sufficient number of nucleotides, generally 3.2.12 PCR buffer, n—a liquid medium that provides the
20 to 35, to facilitate specific hybridization with the amplified appropriate salts, cofactors, and pH value required for ampli-
DNA product, and may be labeled with a detection molecule.
fication of DNA by PCR.
It is common to use probes that can bind to the amplified DNA
3.2.13 PCR product, n—the amplified DNA synthesized by
product at positions located between annealing sites of PCR
DNA polymerase in a PCR. This term is sometimes called
primers. In some procedures, however, the probe can be used
amplicon.
as the PCR primer.
3.2.14 primer, n—for PCR, an oligonucleotide of defined
length, generally 20 to 30 bases, complementary to one strand
3 of DNA from the target sequence of interest. A primer pair
Available from the National Committee for Clinical Laboratory Standards, 771
E. Lancaster Avenue, Villanova, PA 19085. defines the segment of the target DNA to be amplified. Primers
E 1873 – 97 (2002)
can be labeled. For RT-PCR, a primer can be an oligonucle- isolated from bacteria that colonize hot environments. Taq
otide of defined length complementary to an RNA target polymerase is a thermostable enzyme commonly used in PCR.
sequence of interest, a random hexamer or oligo deoxythymi-
dine. 4. Significance and Use
3.2.15 primer-dimer, n—in PCR, the extension and ampli-
4.1 This guide is intended for use in any laboratory utilizing
fication of primer pairs (independent of target DNA), usually
PCR or RT-PCR to amplify and detect a specific nucleic acid
caused when there is some complementarity between the two
sequence.
primers at the 38 ends. These form products that are usually the
4.2 The criteria used for evaluation of the amplification
length of the total of the two primers minus the overlap. Since
reactions should be administered by an individual trained in the
they are relatively small and are complementary to the free
use of molecular biological techniques associated with PCR.
primers, they can amplify efficiently and compete with and
hinder amplification of the target DNA in PCR.
5. Principle of the Method
3.2.16 reaction tubes, n—for PCR, tubes that can be heated
5.1 The PCR is an in vitro method that enables the ampli-
to 100°C and cooled to 4°C without any damage resulting in
fication and subsequent detection of segments of nucleic acid
leakage of contents. They should allow a rapid and homoge-
sequences. The increase in amount (amplification) of target
neous transfer of heat from the instrument heating block to the
DNA occurs by an enzyme with DNA polymerase activity in
reaction mix to occur during the PCR procedure. They should
the presence of two, or more, oligonucleotide primers, deoxy-
not be made with substances known to be inhibitory to PCR
++
nucleoside triphosphates, Mg and a defined reaction buffer.
(2,9,10).
Amplification of the DNA follows a reaction cycle that consists
3.2.17 reverse transcription–PCR, RT-PCR, n—an in vitro
of the following.
laboratory method for using RNA as the target molecule for
5.1.1 Heating the reaction mixture to a temperature suffi-
amplification (3,11). A cDNA copy of the RNA target is made
cient to denature the two strands of the target DNA.
by annealing a complementary DNA oligonucleotide to the
5.1.2 Cooling the reaction mixture to a temperature that
RNA target followed by the enzymatic extension of the
allows the primers to anneal with the target DNA strands.
annealed primer by an enzyme with reverse transcriptase
5.1.3 Controlling at a temperature that allows extension of
activity. The cDNA:RNA hybrid is then heat denatured and a
the annealed primers by DNA polymerase to occur on both
complementary primer anneals to the cDNA, and a second
single strands.
DNA strand is synthesized by an enzyme with DNA poly-
5.1.4 The preceding steps (5.1.1-5.1.3) constitute a cycle.
merase activity. The increase in amount (amplification) of the
The cycle is repeated, (for example, 30 times), providing for
cDNA segments occurs during repeated cycles of heat dena-
repeat amplification of the target and any amplified material
turation of the double-stranded DNA, annealing of the primers
produced in the previous cycles. Some amplification protocols
to their complementary DNA strands, and enzymatic extension
lack a discrete extension step, and extension occurs while the
of the annealed primers by DNA polymerase (see 3.2.11). The
reaction mixture is being heated or cooled between the
amplification (of DNA) results in a near exponential increase in
annealing temperature and the denaturation temperature.
the amount of the original RNA segment defined by the
5.2 The RT-PCR is an in vitro method that enables the
antisense primer.
amplification and subsequent detection of cDNA reverse-
3.2.18 reverse transcription/polymerase enzyme, n—for RT-
transcribed from segments of RNA. The replication of RNA
PCR, a single enzyme that mediates reverse transcription of
segments occurs in two steps. A cDNA copy of the original
RNA to cDNA as well as amplification of cDNA by PCR. The
RNA target is made by an enzyme with reverse transcriptase
enzyme rTth is an example of a thermostable DNA polymerase
activity in the presence of a reverse transcription oligonucle-
that can be used to reverse transcribe RNA efficiently in the
otide primer, deoxynucleoside triphosphates, a divalentcation
++
presence of Mn at elevated temperature, and then subse-
and a defined reaction buffer. In the second step the amount of
quently act as a DNA polymerase to amplify the cDNA target
cDNA is increased (amplified) by PCR. The increase in amount
in a single tube. Alternatively, two enzymes may be used, one
(amplification) of cDNA is catalyzed by an enzyme with DNA
a reverse transcriptase which transcribes the
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