Nucleic acid amplification technologies

Nucleic acid amplification technologies Richard D. Abramson and Thomas W. Myers

Roche Mo lecu la r Systems Inc., Alameda, USA

The polymerase chain reaction, Q~ replicase methodology, the ligase chain reaction, the self-sustained sequence replication system, and the new strand displacement assay have continued to progress with the development of improved reagents and new applications. These advances in enzymatic nucleic acid amplification strategies continue to provide research and medical communities with an ever-improving arsenal of ways to amplify RNA

and DNA.

Current Opinion in Biotechnology 1993, 4:41--47

Introduction New PCR reagents

The ability to isolate and manipulate DNA sequences has revolutionized the biological sciences. With the introduction of recombinant DNA technology, the study of the structure and function of individual genes has progressed rapidly. In the last decade, major advances in the study of specific nucleic acid sequences have oc- curred with the advent of the polymerase chain reaction (PCR) and to a more modest extent by other in vitro DNA amplification technologies (Table 1). Cell- free gene amplification has simplified many of the stan- dard procedures for cloning, analyzing and modifying nucleic acids. DNA fragments from complex genomes can be obtained in a matter of hours, and even a single molecule can be sufficient as starting material. The sim- plicity of nucleic acid amplification, as well as its speed and sensitivity, makes it ideally suited to a wide variety of applications, including the diagnosis and characterization of infectious and genetic diseases (including cancer), forensic analysis, environmental microbiology, evolutionary studies, and basic research.

The polymerase chain reaction

The polymerase chain reaction (PCR) method of in vitro DNA amplification is based on annealing and extending two oligonucleotide primers that flank the target region of duplex DNA [1,2]. If the newly synthesized strand extends to or beyond the region comple- mentary to the other primer, it can serve as a template for a subsequent primer extension reaction. Conse- quently, repeated cycles result in the exponential ac- cumulation of a discrete fragment with termini defined by the 5' ends of the primers.

The first advance in PCR technology came with the introduction of a thermostable DNA polymerase, Ther- mus aquaticus DNA polymerase (Taq pol) [3]. Use of a thermostable enzyme not only simplified the procedure but increased the specificity and yield of the reaction. Recent developments in PCR protocols and reagents have affected critical parameters, including the misincorporation rate, specificity, and maximum length of PCR products. Taq pol lacks 3' to 5' exonuclease activity, but has a 5' to 3' exonuclease activity. Ge- netically engineered variants of Taq pol lacking 5" to 3' exonuclease activity have recently been described (RD Abramson and DH Gelfand, Abstracts of the 92nd Gen- eral Meeting of the American Society for Microbiology, New Orleans, May 1992, p 200) [4,5]. One such variant, Stoffel Fragment [4], exhibits a broader Mg2+ optimum, a lower processivity, and a higher thermostability (RD Abramson, S Stoffel, P Landre, unpublished data). The broader Mg 2+ optimum is useful for performing mul- tiplex PCR, whereas the lack of 5' to 3' exonuclease activity and increased thermostability enhances the amplification of G+C-rich targets. The absence of 5' to 3' exonuclease activity has also proved valuable when a large amount of specific product is desired (RK Saiki, personal communication). Lower processivity decreases mismatch extension efficiency [6], making the enzyme useful for the amplification of rare mutant alleles in a background of normal DNA, using allele- specific primers where suppression of 3' mismatch extension is desired. Suppression of mismatch extension may also enhance the fidelity of the PCR [5,6].

Several thermostable DNA polymerases possessing 3' to 5' exonuclease proofreading activity have been described (FC Lawyer and DH Gelfand, Abstracts of the 92nd General Meeting of the American Society for Mi- crobiology, New Orleans, May 1992, p 200) [7,8,9°]. The

Abbreviations LCR--ligase chain reaction; PCR--polymerase chain reaction; pol--polymerase; Q~R--Q[3 replicase; RT--reverse transcriptase;

SDA--strand displacement amplification; 3SR--self-sustained sequence replication.

© Current Biology Ltd ISSN 0958-1669 41

42 Analytical biotechnology

Table 1. Comparison of enzymatic nucleic acid amplification strategies,

Number of Amplification target-specific Level of

Technique characteristic a Enzyme(s) Temperature oligonucleotides amplification

PCR Target Thermostable 50-98 °C 2 1012 DNA polymerase thermal cycling

QISR Probe Q~ replicase 37 °C 1 109 (4 subunits) isothermal

LCR Probe Thermostable 50-98 °C 4 105 DNA ligase thermal cycling

3SR Target Reverse 42 °C 2 1010 transcriptase isothermal

RNase H RNA polymerase

SDA Target DNA polymerase b 37 °C 4 10 7

Restriction isothermal endonuclease

aTarget amplification provides additional sequence information whereas probe amplification provides no sequence information beyond oligonucleotides; bDNA polymerase must lack the 5' to 3' exonuclease activity. LCR, ligase chain reaction; PCR, polymerase chain reaction; Q[gR, Q[8 replicase; SDA, strand displacement amplification; 3SR, self-sustained sequence replication.

presence of a 3' to 5' exonuclease activity is expected to lower the misincorporation rate of a polymerase. Fi- delity studies indicated an error rate for Thermococ- cus litoralis DNA polymerase 2-4 times lower than the respective values obtained using enzymes without proofreading activity [7,8]. Similarly, the error rate for Pyrococcus furiosus DNA polymerase was shown to be more than 10-fold lower [9"]. Fidelity, however, is affected not only by the exonuclease properties of the polymerase, but also the reaction conditions [10"]. Thus, for Taq pol, a greater than 70-fold difference in base substitutions was seen by varying the MgC12 concentration, with the error rate under high fidelity conditions approaching or exceeding that reported for proofreading enzymes [10"]. The presence of a 3' to 5' exonuclease can introduce new limitations to the PCR. The ability of the exonuclease to degrade primers from the 3" end can impair the correct functioning of the primers, resulting in either a lower yield or non- specific products [11"']. This may be attenuated by a single phosphorothioate bond at the 3' terminus of the primer [11"q. Whether a phosphorothioate bond will also protect a mismatched base at the 3" end, allowing sequence-specific extension reactions, has yet to be demonstrated. An additional complication of the presence of a proofreading exonuclease activity arises in the amplification of genomic DNA [7], where a large number of 3' ends are present to sequester the enzyme non-productively.

A thermostable DNA polymerase closely related to Taq pol, Thermus thermophilus DNA polymerase ( Tth pol), has been found to possess efficient reverse transcrip-

tase (RT) activity in the presence of MnC12 [12"]. Many of the problems associated with the high degree of RNA secondary structure can be minimized by performing reverse transcription at elevated temperatures. After chelation of the MnC12 and addition of MgCl2, the Tth pol can be used subsequently for PCR amplification of the newly synthesized cDNA. More recently, it was found that by modifying reaction parameters, there was no need to alter the reaction buffers for the two enzymatic steps (KKY Young, RM Resnick, 2XV Myers, 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, September 1991, p 182) [13].

New procedures for increased PCR specificity and efficiency

New approaches to improve reaction specificity have been developed. These strategies are based on the recognition that thermostable DNA polymerases re- tain considerable activity at low temperatures allowing extension of pr imer- template complexes that are not perfectly matched ('mis-priming'), as well as the amplified extension of two primers across one another 's sequences (primer dimerization). One method to avoid such events involves delaying the complete mixture of all PCR reactants, and thus the start of the reaction, until they have been heated to a temperature that prevents primer annealing to non-targeted sequences [14]. This manual 'hot-start ' method often increases amplification efficiency and specificity. A technique devised recently

Nucleic acid amplification technologies Abramson and Myers 43

to automate the hot-start process involves the use of a solid wax layer, providing a simple mechanism for synchronizing hot-start amplifications without manual intervention [15"]. An enzymatic method of automated hot-start PCR has also been described (S Kwok et al. and S Kinard et al., Abstracts of the 92nd General Meet- ing of the American Society for Microbiology, New Or- leans, May 1992, p 116). This procedure involves gen- erating dU-containing amplification products by sub- stituting dUTP for dTTP in the PCR. Treatment with uracil-N-glycosylase at elevated temperatures immedi- ately before thermal cycling cleaves any dU-containing extension products that were the result of low-temperature mis-priming. The addition of Escherichia coli single-strand DNA-binding protein has also been reported to increase specific amplification [16,P1"]. Pre- sumably, this is another form of protein-mediated hot- start PCR, where the single-strand DNA-binding protein binds the primers and template at low temperatures, preventing mis-priming and primer dimerization.

Novel PCR strategies and applications

A variety of useful strategies have been implemented to expand the applications of PCR. These include gene walking by the use of a specific target primer and a panel of random oligonucleotide primers [17], the gen- eration of a panhandle-shaped template which places a known DNA sequence on the uncharacterized side of the sequence of interest [18], and a variant of ligation- mediated PCR whereby a sequence-specific 'adapter- tag' provides one target for primer annealing, and a universal 'bubble-tag' provides the second target for primer annealing [19].

A recently developed detection assay exploits the 5' to 3' exonuclease activity of Taq pol to generate a specific signal concomitantly with amplification [20,21]. A labeled oligonucleotide probe is introduced into the reaction, such that during amplification Taq pol de- grades the probe. The assay is sensitive and specific and is a significant improvement over more cumber- some hybridization-based methods. A homogeneous assay for PCR product detection was demonstrated based upon the increased fluorescence that ethidium bromide exhibits when bound to double-stranded DNA [22",P2]. The fluorescence is measured by direct excita- tion through the wails of the amplification tube before and after amplification, or continuously during amplification. Although simultaneous amplification and detection of product within the tube has many advantages, this method is not as specific as probe hybridization methods.

Advances in PCR applications have been seen in a variety of fields. These include environmental microbiology, where PCR amplification has been used to detect microorganisms in food, water, soil, and sed- iment samples [23-25]. More recently, in the field of prenatal diagnosis of genetic disease, PCR was used

for the first preimplantation diagnosis of cystic fibro- sis after in vitro fertilization [26-q.

Q~ replicase

A different approach to the in vitro amplification of either DNA or RNA has been achieved by a probe amplification method. Recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by the RNA-directed RNA polymerase Q~ replicase (Q~R) have been constructed [27]. While the ability to utilize either RNA or DNA targets is advantageous and avoids a RT step, double- stranded templates must be denatured first. As QI3R is used to amplify a probe signal rather than the target, non-specifically bound or unhybridized probe must be completely eliminated. Additionally, as only the probe is amplified, no new sequence information can be obtained. The development of a method for reversible target capture [28] has significantly lowered background noise. In the presence of excess Q~R, each molecule of hybridized probe gives rise to a single-stranded RNA product. As both the parent and daughter strands can serve as the template, exponential amplification pro- ceeds such that a single template can ideally produce 1012 replicates in less than 15 minutes [29"]. Theoreti- cally this amplification system is amenable to quanti- tation because during exponential synthesis the dou- bling time is constant [30]. The expression and purification of a recombinant Q~R [31,P3"] should allow for faster development of this technology by increasing the availability of reagents, lowering reagent costs, and reducing levels of contaminating bacteriophage RNA [32,33]. Further work defining requirements for replicatable RNA reporters [34,P4 o] will undoubtedly lead to increased use of Q~R [31], although applications re- quiring allelic discrimination are not readily amenable to this amplification technique.

Ligase chain reaction

Another method of probe amplification is the ligase chain reaction (LCR). LCR is based on the gene detection technique of Whiteley et a t (Applied Biosystems) [P5] and Landegren et al. [35], which utilizes the ability of a DNA ligase to join covalently two oligonucleotides that are adjacent to each other on a nucleic acid target. The assay is readily amenable to automation and detection [35,P5]. Further refinement of this technique resulted in either linear or exponential amplification depending upon whether sequential rounds of ligation following denaturation were performed with a single pair or two pairs of oligonucleotides [36]. Unlike PCR, this process amplifies regions of target that contain only the sequence of the oligonucleotides. A severe limitation to this method is the requirement for fresh ligase following each denaturation, a problem that Wu


and Wallace [36] suggested would be overcome by using a thermostable ligase. The cloning and expression of a thermostable ligase [37",38"], in addition to alter- ations in reaction conditions [39,40"], have allowed for the detection of a single base substitution with a sensitivity of 200 target molecules. The combination of a primary amplification system, such as PCR, with a secondary LCR amplification/detection has been suggested to allow screening of genetic diseases and/or viral variants containing multiple mutations [40"]. Al- though the discriminatory power of LCR has been suggested to be quite good, contamination issues have yet to be resolved [41] and low level blunt-end ligation may generate background problems.

Self-sustained sequence replication system

Kwoh et al. [42] have devised a transcription amplification system similar to the nucleic acid sequence-based amplification system [43,P6"], which consists of performing cDNA synthesis with either an RNA or DNA target using oligonucleotides containing RNA polymerase promoter regions at the 5' terminus, to produce one copy of a double-stranded DNA template. After denaturation and further cDNA synthesis by RT, the cDNA template is then transcribed by an RNA polymerase. The multiple copies of RNA produced can serve as target sequences for additional cycles. The inclusion of a third protein, RNase H, obviated the necessity to heat denature the template following RT [44]. This isothermal self-sustained sequence replication (3SR) system achieved a 107-fold amplification in less than two hours at 37 °C. Additionally, unless a heat denaturation step is specifically added prior to RT, the reactions performed at 37 °C would theoretically pre- clude amplification of double-stranded DNA allowing preferential amplification of RNA, providing the nucleic acid preparation was free of single-stranded DNA. This attribute of the 3SR technique has been used to analyze and detect mutations in zidovudine-resistant HIV [45"] and for the detection of HIV RNA in pediatric patients [46"]. Further optimization of reaction conditions sug- gests that the RNase H activity of avian myeloblastosis virus RT is enhanced in 10% dimethyl sulfoxide and a neutral polyalcohol [47"',P7"], allowing the E. coli RNase H to be omitted. Issues such as the fidelity of the reverse transcriptase, length limitations on amplification, and contamination prevention, have yet to be resolved.

Strand displacement amplification

Strand displacement amplification (SDA) is an isothermal procedure that exploits the ability of a restriction enzyme to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and for a DNA polymerase to extend the 3'-hydroxyl group at

the nick and displace the downstream DNA [48"]. Expo- nential amplification is achieved by coupling sense and antisense reactions. The choice of restriction enzyme is limited because the enzyme must be capable of nicking a hemiphosphorothioate recognition site and the poly- merization/displacement step also must regenerate a site that is capable of being nicked by the restriction enzyme. Furthermore, an additional limitation is that the target sequence must not contain a recognition site for the restriction enzyme used in the SDA. Walker et al. [48"1 achieved a 106-fold amplification and a sensitivity of approximately 100 copies of a 47-base-pair target DNA fragment in the genome of Mycobacterium tuberculosis. A major drawback of this method was that it required the DNA sample to be digested with a restriction enzyme prior to SDA in order to generate a target fragment with defined 5'- and 3'-ends. There- fore, the target generally had to be double-stranded DNA and the target sequence had to be flanked by the appropriate recognition sites.

This limitation has been circumvented by adding two additional primers which lack the SDA restriction enzyme recognition sequence [49"'1. The new scheme achieved a 107-fold amplification and a claimed sensitivity of approximately 10-50 copies of M. tuberculosis. Alteration of reaction conditions allowed for as much as 10btg of non-target DNA to be present in the SDA, although higher levels of input target (104 copies) were used. Several difficulties still exist in this amplification technique: a preliminary RT step is required for RNA amplification; polymerase extension under non-stringent hybridization conditions significantly compromises analytical detection limits, particu- larly at modest concentrations of non-target DNA; the target length is limited by the efficiency of displacement synthesis by the DNA polymerase and there is an approximately 10-fold reduction in amplification for each 50 nucleotide increase in target size [49"q; the prob lem of 'carryover' amplification products was noted in both reports of this technique, possibly exac- erbated by the necessity of multiple reagent additions, and will need to be resolved before the technique is put to widespread use in clinical diagnostics.

Conclusion

The development of new and/or improved nucleic acid amplification reagents and methodologies has continued at a rapid rate, primarily as a result of the fast pace of basic and clinical research in the scientific com- munity and the potential rewards in the commercial world [50",51",Pl",P2,P3",P4",P5,P6",P7"',P8"]. While the well known PCR continues to dominate the research and diagnostic fields, alternative diagnostic schemes are being pursued rigorously. Although the primary impetus for expanding the amplification repertoire ap- pears to be commercially driven, these new techniques are also being used to complement the PCR, and are more recently being used in conjunction with the PCR


to a u g m e n t its app l i c a t i ons . I m p o r t a n t t e c h n i c a l i s sues n o w r e s o l v e d f o r t he PCR s u c h as sensi t ivi ty , s p e c i -

ficity, r o b u s t n e s s , a u t o m a t i o n o f r e a c t i o n s a n d d e t e c - t ion , as we l l as c o n t a m i n a t i o n i s sues , h a v e fo r t h e m o s t

p a r t y e t to b e a d d r e s s e d fo r t h e a l t e rna t ive s t ra teg ies . T h e u l t ima te f ru i t i on o f all t h e a m p l i f i c a t i o n t e c h n o l o - g ies wi l l resu l t in t he i r w i d e s p r e a d i m p l e m e n t a t i o n in to s u c h d i v e r s e f i e lds as m o l e c u l a r b io logy , g e n e t i c s , mi- c r o b i o l o g y , d i a g n o s t i c s , a n d fo r ens i c s .

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest • . of outstanding interest

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2. MULLIS KB, FALOONA FA: Specific Synthesis o f DNA In Vitro Via a Polymerase-catalyzed Chain Reaction. Meth- ods Enzymol 1987, 155:335-350.

3. SAIKI RK, GELFAND DH, STOFFEL S, SCHARF SJ, HIGUCHI R, HORN GT, MULLIS KB, ERLICH HA: Prinxer-directed Enzy- matic Amplification of DNA with a T h e r m o s t a b l e DNA Polylnerase. Science 1988, 239:487-491.

4. ABRAMSON RD, HOLLAND PM, WATSON R, GELFAND DH: Characterization o f the 5' to 3' E x o n u c l e a s e Activity o f Thermus aquat icus DNA Polyxnerase. FASEBJ 1991, 5:A437.

5. BARNES WM: The Fidelity of Taq Polymerase Catalyz- ing PCR is Improved by an N-Terminal Dele t i on . Gene 1992, 112:29-35.

6. HUANG M-M, ARNHEIM N, GOODMAN MF: E x t e n s i o n o f Base Mispairs by Taq DNA P o l y m e r a s e : I m p l i c a t i o n s for Single N u e l e o t i d e D i s c r i m i n a t i o n in PCR. Nucleic Acids Res 1992, 20:4567~4573.

7. CARIELLO NF, SWENBERG JA, SKOPEK TR: Fidelity o f Ther- mocoecus l i toralis DNA Polytnerase (Vent TM) in PCR Determined by Denaturing Gradient Gel Electrophore- sis. Nucleic Acids Res 1991, 19:4193-4198.

8. MATI'ILA P, KORPELA J, TENKANEN T, PITKMX!EN K: Fidelity o f DNA S y n t h e s i s b y t h e Thermococcus litoralis DNA Polyxnerase - - an E x t r e m e l y Heat Stable En- zynxe with Proofreading Activity. Nucleic Acids Res 1991, 19:4967-4973.

9. LUNDBERG KS, SHOEMAKER DD, ADAMS MWW, SHORT JM, SORGE JA, MATHUR EJ: High-fidelity Amplification Us- i n g a T h e r m o s t a b l e DNA Polynlerase Isolated f r o m Pyrococcus f u r i o s u s . Gene 1991, 108:1-6.

This paper presents a characterization of the thermostable proofreading DNA polymerase isolated from Pyrococcus furiosus (Pfu). It provides an evaluation of the 3' to 5' exonuclease activity and polymerase fidelity of Pfu DNA polymerase during PCR amplification.

10. ECKERT KA, KUNKEL TA: The Fidelity ofDNA Polylnerases • . Used in the Polymerase Chain React ion . In Polymerase

Chain Reaction: A Practical Approach, reprinted edn (with corrections). Edited by McPherson MJ, Quirke P, Taylor GR. New York: Oxford University Press; 1992: 225-244.

An excellent discussion of DNA polymerase fidelity and its role in PCR amplification. Includes a detailed description of the variables

that influence Taq DNA polymerase fidelity, and their relevance to high-fidelity synthesis.

11. SKERRA A: P h o s p h o r o t h i o a t e P r i m e r s I m p r o v e t h e Am- .. p l i f i cat ion o f DNA S e q u e n c e s b y DNA P o l y m e r a s e s

with Proofreading Activity. Nucleic Acids Res 1992, 20:3551-3554.

This manuscript illustrates the resistance of phosphorothioate link- ages towards the 3' to 5' exonudease activity of two thermostable DNA polymerases, allowing for increased efficiency and specificity of PCR amplification with a proofreading DNA polymerase. It is especially important for applications where high-fidelity synthesis is of the utmost importance.

12. MYERS TW, GELFAND DH: R e v e r s e Transcr i p t i on a n d DNA Atnplification by a Thermus thermophilus DNA Poly- merase. Biochemistry 1991, 30:7661-7666.

The first description of efficient thermostable RT activity. Tth DNA polymerase is shown to be 100-fold more efficient than Taq DNA polymerase in a coupled RT/PCR, with a sensitivity of target detection as low as 100 copies of synthetic cRNA or 80 picograms of total cellular RNA.

13. MYERS TW, GELFAND DH: Enzymatic Properties of a DNA P o l y m e r a s e f r o m Thermus thermophilus o n RNA and DNA Templates . J Cell Biochem 1992, Supplement 16B:29.

14. MULLIS KB: The Polymerase Chain Reaction in an Ane- mic Mode: How to Avoid Cold Oligodeoxyribonuclear Fusion. PCR Methods Applic 1991, 1:1-4.

15. CHOU Q, RUSSELL M, BIRCH DE, RAYMOND J, BLOCH W: Pre- v e n t i o n o f Pre-PCR Mis-primirtg a n d Primer Dimeriza- t ion I m p r o v e s L o w - c o p y - n u m b e r Ampl i f i ca t ions . Nu- cleic Acids Res 1992, 20:1717-1723.

A detailed discussion of hot-start PCR and how it can improve the specificity, yield, and precision of amplifying DNA, especially at low copy number in a high background of complex DNA. The inclusion of a wax vapor barrier simplifies the technique, making it readily synchronized and automated.

16. CHOU Q: Minimizing D e l e t i o n Mutagenes i s Artifact Dur- ing Taq DNA Polyxnerase PCR by E. coli SSB. Nucleic Acids Res 1992, 20:4371.

17. PARKER JD, RABINOVITCH PS, BURMER GC: Targeted Gene Walking Poly'tnerase Chain React ion . Nucleic Acids Res 1991, 19:3055-3060.

18. JONES DH, WINISTORFER SC: Sequence Specific Genera- t i o n of D N A Panhandle Permits PCR Anapli f ieat ion o f Unknown Flanking DNA. Nucleic Acids Res 1992, 20:595-600.

19. SMITH DR: Ligation-tnediated PCR of Restriction Frag- m e n t s f rom Large DNA Molecules. PCR Methods Applic 1992, 2:21-27.

20. HOLLAND PM, ABRAMSON RD, WATSON R, GELF&ND DH: De- tection of Specific Polymerase Chain Reaction Product by Utilizirtg t h e 5' to 3' E x o n u c l e a s e Act ivi ty o f Ther- m u s aquat icus DNA Poly~merase. Proc Natl Acad Sci USA 1991, 88:7276-7280.

21. HOLLAND PM, ABRAMSON RE), WATSON R, WILL S, SAIKI RK, GELFAND DH: D e t e c t i o n o f Spec i f i c Polymerase Chain React ion Product by Utillzitlg the 5' to 3' Exonuclease Activ i ty o f Thermus aquat icus DNA P o l y m e r a s e . Clin Chem 1992, 38:462--463.

22. H1GUCHI R, DOLLINGER G, WALSH PS, GRIFFITH R: Sitnul.- taneous Atnp l i f i ca t i on and D e t e c t i o n o f Speci f ic DNA Sequences. Biotechnology 1992, 10:413~i17.

This paper describes a simple method for simultaneously amplifying a specific DNA sequence and detecting the product of the amplification in a truly homogeneous fashion. This assay allows for the continuous monitoring of PCR amplification during thermal cycling. The method can be easily automated.

23. WERNARS K, DELFGOU E, SOENTORO PS, NOTHERMANS S: Suc- cessful A p p r o a c h for D e t e c t i o n o f Low N u m b e r s of


Enterotoxigenic E s c b e r i c b i a coil i n Minced Meat by Using the Po lymerase Cha in Reaction. Appl Environ Microbiol 1991, 57:1914-1919.

24. BEJ AK, MAHBUBANI MH, DICESARE JL, ATLAS RM: Poly- merase Cha in React ion-gene Probe Detect ion o f Micro- o r g a n i s m s by Using Fi l ter-concentrated Samples. Appl Environ Microbiol 1991, 57:3529-3534.

25. TSAI Y-L, OLSON BH: Detect ion of Low N u m b e r s o f Bacte- rial Cells in Soils and Sediments by Po lymerase Chain Reaction. Appl Environ Microbiol 1992, 58:754-757.

26. HANDYSIDE All, LESKO JG, TARiN JJ, WINSTON RML, HUGHES • . MR: Bir th o f a Normal Girl After I n Vi tro Fertilization

and Pre implan ta t ion Diagnostic Test ing for Cystic Fi- brosis . N Engl J Med 1992, 327:905-909.

An interesting use of PCR amplification for prenatal diagnosis of in- herited diseases. Preimplantation diagnostics allows parents to be certain before pregnancy that their offspring is free of a specific genetic disorder.

27. LIZARDI PM, GUERRA CE, LOMELI H, TUSSlE-LUNA I, KRAMER FR: Exponen t i a l Amplif icat ion o f Recombinant-RNA Hybridizat ion Probes. Biotecbnology 1988, 6:1197-1202.

28. MORRISSEY DV, LOMBARDO M, ELDREDGE JK, KEARNE¥ DR, GROODY EP, COLLINS ML: Nucleic Acid Hybridizat ion As- says Employ ing dA-Tailed Capture Probes. I. Multiple Capture Methods. Anal Biochem 1989, 181:345-359.

29. CAHILL P, FOSTER K, MAHAN DE: Polyxnerase Cha in Reac- t ion an d Q~ Replicase Amplif icat ion. Clin Chem 1991, 37:1482-1485.

A short review comparing and contrasting the PCR and Q~ replicase amplification techniques. The authors from Gene-Trak Systems provide a good discussion on technical details as well as on the im- plications of sample processing for each of the two methodologies.

30. LOMELL H, TYAGI S, PPaTCHARD CG, LIZARDI PM, KRAMER FR: Quantitat ive Assays Based o n the Use o f Replicatable Hybridizat ion Probes. Clin Chem 1989, 35:1826-1831.

31. I~ZARDI PM, KRAMER FR: Exponen t ia l Ampli f icat ion o f Nucleic Acids: N e w Diagnost ics Using DNA Poly- merases and RNA Repl l cases . Trends Biotechnol 1991, 9:53-58.

32. MUNISHKIN AM, VORONIN LA, UGAROV VI, BONDAREVA LA, CHETVERINA H¥, CHETVERIN AB: Efficient T e m p l a t e s for Q~ Repliease are F o r m e d by Recombina t ion f r o m Heterologous Sequences. J Mol Biol 1991, 221:463~172.

33. CHETVERIN AB, CHETVERINA HV, MUNISHKIN AV: On the Na- ture of Spon taneous RNA Synthesis by Q~ Replicase. J Mol Biol 1991, 222:3-9.

34. AXELROD VD, BROWN E, PRIANO C, MILLS DR: Coliphage Q~ RNA Repl icat ion: RNA Catalytic for Single-strand Release. Virology 1991, 184:595-608.

35. LANDEGREN U, KAISER R, SANDERS J, HOOD L: A Ligase- media ted Gene Detect ion Technique. Science 1988, 241:1077-1080.

36. Wu DY, WALLACE RB: The Ligation Aamplification Reac- t ion (LAR) - - Ampl i f ica t ion o f Specific DNA Sequences Using Sequential R o u n d s o f T e m p l a t e - D e p e n d e n t Lig- at ion. Genomics 1989, 4:560-569.

37. LAUER G, RUDD EA, McKAY DL, ALLY A, ALLY D, BACKMAN KC: Cloning, Nucleotide Sequence, and Engineered Expres s ion o f Thermus thermophilus DNA Ligase, a Homolog of E s c h e r i c b i a coli DNA Ligase. J Bacteriol 1991, 173:5047-5053.

The first publication detailing the cloning and expression of a thermostable DNA ligase. The authors from BioTechnica provide the nucleotide sequence and deduced amino acid sequence of the Thermus thermophilus DNA ligase and analyze its sequence similarity with that of other ligases. A partial purification scheme is also presented.

38. BARANY F, GELFAND DH: Cloning, O v e r e x p r e s s i o n and Nucleotide Sequence o f a T h e r m o s t a b l e D N A Ligase- encod ing Gene. Gene 1991, 109:1-11.

A second publication describing the cloning and expression of a thermostable DNA ligase from Thermus thermophilusby genetic comple- mentation. A detailed purification protocol is provided as well as a comparison of codon usage in Thermus aquaticus DNA polymerase I and Thermus thermophilus DNA ligase.

39. BARANY F: Genetic Disease Detect ion and DNA Ampli- f icat ion Using Cloned The rmos tab l e Hgase. Proc Nail Acad Sci USA 1991, 88:189-193.

40. BARANY F: T h e Ligase Cha in Reaction in a PCR World. • . PCR Methods Applic 1991, 1:5-16. An extensive review that provides a detailed description and history of ligases in general, the use of ligases in LCR, and future prospects of LCR. The author also discusses techniques which involve both LCR and PCR.

41. BIRKENMEYER LG, MUSHAHWAR IK: DNA Probe Anaplifica- t ion Methods. J Virol Methods 1991, 35:117-126.

42. KWOH DY, DAVID GR, WFIITFIELD KM, CHAPPELLE HL, DIMICHELE LJ, GINGERAS TR: Transcr ip t ion-based Ampli- f icat ion Sys tem and Detect ion o f Ampli f ied H u m a n Immunode f i c i e nc y Virus Type 1 wi th a Bead-based Sandwich Hybridizat ion Format . Proc N a g Aca d Sci USA 1989, 86:1173-1177.

43. COMPTON J: Nucleic Acid Sequence-based Axnpliflcation. Nature 1991, 350:91-92.

44. GUATELLI JC, WHITFIELD KM, KWOH DY, BARRINGER KJ, RICHMAN DD, GINGERAS TR: Iso thermal , In V i t ro Atnpli- f icat ion o f Nucleic Acids by a Mult ienzytne Reaction M o d e l e d After Retroviral Replication. Proc Natl Acad Sci USA 1990, 87:1874-1878.

45. GINGERAS TR, PRODANOVICH P, LATIMER T, GUATELLI JC, RICHMAN DD, BARRINGER KJ: Use o f Self-sustained Se- quence Replicat ion Amplif icat ion React ion to Ana~ lyze a nd Detect Mutat ions in Zidovudine-res is tant H u m a n Immunode f i c i e nc y Virus. J Infect Dis 1991, 164:1066-1074.

This paper describes the use of 3SR and differential bead-based sandwich hybridization to detect and analyze HIV-1 RNA from infected peripheral blood mononuclear cells and viral stocks obtained after coculture with uninfected lymphocytes. The genotype of the sam- pies was correlated with drug resistance and the PCR and Southern hybridization results of a previous study.

46. BUSH CE, I)ONOVAN RM, PETERSON W'R, JENNINGS MB, BOLTON V, SHERMAN DG, VANDEN BRINK KM, BENINSIG LA, GODSEY JH: Detection o f H u m a n Immunode f i c i ency Virus Type 1 RNA in P lasma Samples f r o m High-risk Pediatric Patients by Using the Self-sustained Sequence Replicat ion Reaction. J Clin Microbiol 1992, 30:281-286.

This publication uses 3SR to amplify HIV-1 RNA in high-risk pediatric patients. Bead-based sandwich hybridization and rare earth metal chelate time-resolved fluorescence were used for detection of the amplified RNA product. The sensitivity of the combined technique was determined to be less than 12 HIV-1 copies with an amplification level of 1010-fold from purified RNA.

47. FAHY E, KWOH DY, GINGERAS TR: Self-sustained Sequence • . Replicat ion (3SR): a n I so the rma l Transcr ip t ion-based

Amplif icat ion System Alternative to PCR. PCR Methods Applic 1991, 1:25-33.

An extensive review that provides great detail of the intricate reaction parameters of the 3SR. The authors also present data indicating that by including cosolvents into the 3SR, the addition of exogenous RNase H can be avoided.

48. WALKER GT, LITTLE MC, NADEAU JG, SHANK DD: Isother~ real I n Vi tro Amplif icat ion of DNA by a Restr ict ion Enzyxne/DNA Polymerase System. Proc Natl Aca d Sci USA 1992, 89:392-396.

The first published description of strand displacement amplification. The authors from Becton Dickinson achieved a 106-fold amplification


of a genomic sequence from Mycobacterium tuberculosis in a four- hour isothermal reaction.

49. WALKER GT, FRAISER MS, SCHRAM JL, LITTLE MC, NADEAU JG, • . MALINOWSKI DP: Strand Displacement A m p l i f i c a t i o n - -

a n I s o t h e r m a l In Vitro DNA Anapl i f i ca t ion T e c h n i q u e . Nucleic Acids Res 1992, 20:1691-1696.

This paper extends the original description of SDA by eliminating the requirement for restriction, enzyme cleavage of the target sample prior to amplification. The authors achieved a 107-fold amplification of a genomic sequence from Mycobacterium tuberculosis in a two- hour isothermal reaction. As in other amplification techniques, the use of organic cosolvents was found to increase the str ingency of SDA.

50. WEISS R: Hot Prospect for N e w G e n e Atnpl i f ie r . Science 1991, 254:1292-1293.

An article discussing the LCR technique and its future prospects. The author also provides insight into the potential legal battle for the patent rights to LCR.

51. LEWIS R: PCR's C o m p e t i t o r s a r e Alive a n d Wel l and M o v i n g Rap id ly T o w a r d s C o m m e r c i a l i z a t i o n . Genetic Engineering News 1992, 12:1.

This article provides a brief synopsis of alternative nucleic acid amplification methodologies, as well as the inventors and developers of each of the techniques.

Patents

of special interest • . of outstanding interest

P1. STRATAGENE: A n Improved Method For Hybridiz ing Nu- cleic Acids Using Single-stranded Nucleic Acid B i n d i n g Pro te in . 24/10/89 89US425864 16/5/91 WO9106679A.

This patent application discusses the use of a single-stranded nucleic acid binding protein to decrease non-specific oligonucleotide hybridization, thus increasing the specificity of hybridization and amplification reactions.

P2. TOSOH CORP.: Method for Detecting or Q u a n t i f y i n g Target Nucleic Acid. 31/10/90 90JP-294305 27/5/92 EP- 487218A.

P3. GENE-TRAK SYSTEMS: P u r i f i c a t i o n o f Q~ Replicase. 1992, US 5141857.

This patent describes a method of purifying Q~ replicase from recombinant sources.

P4. CIBA CORNING DIAGNOSTICS CORP.: A tnp l i f l ca t ion o f Nu- cleic Acid Sequences Using Prhners Containing Q Beta Midivariant DNA Sequences. 16/10/90 90US-598269 22/4/92 EP-481704A.

Details the use of recombinant RNA molecules generated from the small RNA template of the virus MDV-1 with Q~ replicase.

P5. APPLIED BIOSYSTEMS INC.: D e t e c t i o n o f Specif ic Sequences in Nucle ic Acids. 1989, US4883750.

P6. CANGENE CORP.: E n h a n c e d Nucleic Acid Atnp l i f i ca t i on Process. 1992, US 5130238.

This patent describes the nucleic acid sequence-based amplification system.

P7. SISKA DIAGNOS~CS INC.: Nucleic Acid A m p l i f i c a t i o n b y • . 2 -Enzyme, Se l f - sus ta ined S e q u e n c e Rep l i c a t i on Using

RNA-dependent DNA Polytnerase Activity, DNA-dependent DNA Polymerase Activity, RNase H Activi ty and DNA-dependent RNA Polymerase Activity. 13/11/90 90US612688 29/5/92 WO920880 A.

This patent application describes the method of isothermal 3SR.

P8. ABBOT1 ~ LABORATORIES: D i a g n o s t i c DNA Probe and DNA P r i m e r for use in Ligase Chain Reaction or Polymerase C h a i n R e a c t i o n DNA Atnp l i f l ca t ion , 28/9/90 90US589948 1/4/92 EP477972A.

This patent applicatlon describes a set of four DNA primers for hu- m a n papilloma virus and theft use in LCR and PCR.

RD Abramson and TW Myers, Program in Core Research, Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, California 94501, USA.

Documents

Nucleic acid amplification technologies