Application of PCR to human gene detection Y-M. Dennis Lo and Alexander F. Markham John Radcliffe Hospital, Oxford, UK The advent of the polymerase chain reaction has stimulated the development of a number of rapid methods for characterizing human genes. In addition, the unprecedented level of sensitivity offered by some of these methods may prove to be of great value in the detection of minority cell populations. Over the past year, technical developments have been made in this area. Current Opinion in Biotechnology 1992, 3:8-11

Introduction Since 1985, a large number of applications based on the potymerase chain reaction (PCR) [1] have been described. This review discusses some of the advances made over the past year in the use of PCR in human gene detection, especially for the diagnosis of known mutations. Other areas of PCR technology not covered by this paper are the subject of a number of recent reviews and books [2--4]. Readers are also referred to reviews in this issue by Cotton (pp 24--30) and l.~adegren (pp 12-17).

Multiplex and double amplification refractory mutation systems Allele-specific PCR [ 5] or the amplification refractory mutation system (ARM) [6] offers a rapid diagnostic method for the detection of mutant or disease susceptibility alleles. The principle that ARMS is based upon relies on the fact that the Taq polymerase commonly used in PCR lacks 3'-5' exonuclease activity and thus a mismatch between a DNA template and the 3' end of a PCR primer will significantly reduce the amplification efficiency. As originally described [5,6], ARM reactions were performed in pairs, one reaction using an ARMS primer against the normal allele and the other one using an ARMS primer against the mutant allele. One drawback of this approach is that the number of parallel reactions increases with the number of allelic variants at a particular locus. Multiplex ARMS offers a solution to this problem and was first developed for HLA genotyping [7]. In multiplex ARMS, a number of ARMS primers, each specific for a particular allele, are present in a single reaction in a single tube. PCR products corresponding to different alleles can

then be distinguished by length [7] or by other physical characteristics, such as different fluorescent labels [8] on different ARMS primers. Multiplex ARMS is most suited to the analysis of genetic loci in which the alleles exhibit sequence variation in different multiple positions, for example, HLA alleles. Hence ARMS primers can be constructed to prime at multiple positions within the target locus thus providing different sized PCR products [7]. The practical considerations that are applicable to conventional multiplex PCR for deletion screening [9] also apply to multiplex ARMS. Thus, it is important for ARMS primers to exhibit allele specificity at a common temperature and have sinailar amplification efficiencies. These problems can generally be solved by adjusting the relative primer concentrations [7], primer lengths and the type of deliberate mismatches at a position 2 or 3 bp from the 3' end [6]. Recently, two groups have presented encouraging data in applying multiplex ARMS to the diagnosis of I]-thalassaemia [P Fortina, G Monokian, R Conant, G Dotti, W Hitchcock, E Rappaport, E Schwartz, S Surrey: abstract B/ood 1991, 78 (suppl 1):194A] and cystic fibrosis [MJ Schwarz, NH Robertson, RM Ferrie, SAVaudin, M Super, S Little abstract AmJHum Genet 1991, 49:186]. A further development in ARMS technology is double ARMS [10.] in which two different allele-specitic ARMS primers are simultaneously used in a single reaction. As each ARMS primer hybridizes at a different polymorphic site, double ARMS allows direct haplotype determination without the need for pedigree analysis and without resorting to single molecule dilution [11], or single sperm typing [12]. Another advantage of double ARMS is that it has a greatly enhanced specificity compared with single ARMS. This may prove useful in the detection of a minority DNA population amongst a background of related but non-identical DNA molecules, for example, for the study of the phenomenon of chimerism following bone marrow transplantation.

Abbreviations A-RFLP--artificial restriction fragment length polymorphism; ARM--amplification refractory mutation system; PCR--polymerase chain reaction; RFLP--restriction fragment length polymorphism;SSCP--single-strand conformation polymorphism.

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Application of PCRto human gene detection Lo and Artificial restriction fragment length polymorphism The detection of a restriction fragment length polymorphism (RFLP), which distinguishes a mutant from a normal allele, was one of the first methods used for analyzing PCR products amplified from genomic DNA [1]. The majority of DNA polymorphisms, however, are not associated with the creation or destruction of restriction sites. Haliassos et al. [13] have shown that when a DNA mutation does not alter a restriction site, such a site can be introduced by a PCR primer with a specially chosen sequence at the 3' end of the primer. Over the past year, this artificial-RFLP (A-RFLP) method has been adopted for the diagnosis of a number of genetic diseases such as 13-thalassaemia [14] and phenylketonuria [15"]. Recently, it has been shown that by using restriction enzymes that recognize several sequences such as Hinfl for which the recognition site is GANTC, very complex allelic systems may be rapidly characterized using A-RFLP [16]. Thus, Patel et al. [16] have applied A-RFLP to divide the 17 HLA DQB1 alleles into those with and those without aspartate at codon 57. This system may be used when screening populations for a susceptibility to insulin-dependent diabetes mellitus [17]. Compared with ARlVlSbased techniques, A-RFLP is less reliant on stringent amplification conditions for discrimination of alleles as this function is performed by the restriction step. Furthermore, A-RFLP-based methods are potentially more robust when amplifying from sources where the exact amount of DNA template is ditficult to quantify accurately, for example, in pamtfin-embedded tissues, ARMS-based technology, however, has the advantage of being more rapid as the restriction step is not necessary.

Single-strand conformation polymorphism analysis Single-strand conformation polymorphism (SSCP) analysis is based on the principle that the mobility of singlestrand DNA in non-denaturing polyacrylamide gels is dependent not only on its length but also on its sequence [18]. It is thought that this phenomenon is due to secondary conformations taken up by single-strand DNA. SSCP analysis has been applied to the diagnosis of phenylketonuria [19] and Tay-Sachs disease [20]. Early protocols for SSCP analysis employed radioactivity, which tends to limit its diagnostic use. Recently, three groups have described non-radioactive methods for SSCP analysis using silver staining [20], ethidium bromide staining [21] and fluorescence [22]. Fluorescent SSCP is performed by using fluorescent PCR primers [8] and has the potential to be automated using a laser scanner. Furthermore, a standard PCR product ladder, with a differently coloured label than the test product and containing all the alleles under study, may be included on each track as a reference to further facilitate the readout process.

Markham

In applying SSCP analysis to the elucidation of multi-allelic systems, the practical difficulty of distinguishing a large number of alleles on a single polyacrylamide gel can be overcome by performing an initial subdivision of a complex allelic series into a number of simpler subgroups using ARMS. Subsequently, those sequences that give positive ARMS reactions are further subtyped using SSCP. This ARIVIS-SSCP strategy has been used for HLA DQB1 typing in which the DQBll alleles are subdivided into 4 ARMS-groups followed by SSCP subtyping [22]. This approach combines the rapidity of ARMS analysis in the initial subdivision with the flexibility of SSCP analysis to detect new alleles in the final typing step and has applications in a number of complex genetic systems, such as HLA class I alleles and the T-cell receptor complex.

Heteroduplex analysis Heteroduplex analysis is a simple method for revealing genetic differences between individuals in which the PCR products from two individuals are mixed, denatured, reannealed and the heteroduplexes formed are detected on a non-denaturing polyacrylamide gel [23]. Heteroduplexes exhibit slower electrophoretic mobility than homoduplexes. This method is most valuable for identifying mutations resulting from insertion or deletion of DNA sequences. Thus, Cai et al. [24] have used this method in the diagnosis of 13-thalassaemia and shown that the technique can detect mutants with a 2-25 bp deletion. Other genetic diseases in which heteroduplex analysis has been applied include Tay-Sachs disease [25] and familial hypercholesterolaemia [26].

PCR in the detection of minority cell populations The exquisite sensitivity of PCR opens up the possibility of detecting cell populations that are present at extremely low frequencies. Smith et al. [27"'] describe an ingenious method for detecting circulating tumour cells in patients with malignant melanoma using reverse transcriptase PCR. The target of the reverse transcriptase PCR is a gene that is only transcribed actively in the tumour tissue (the tyrosinase gene in [27"']). The main advantage of the method is that it does not depend on the complete characterization of cancer-specific genetic abnormalities. The recent availability of a thermostable DNA polymerase from 7bermus tbermopbilus that uses manganese as a metal cation and that is able to synthesize DNA from both RNA and DNA templates can further simplify this method [28*]. Another development in the field of minority nucleic acid population detection is the improvement in nested PCR technology, Nesting is a simple procedure that enhances the sensitivity of the PCR by using two sequential rounds of amplification with an outer and an inner set of primers [29,30]. One potential problem of nesting is that the sam-

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10 Analyticalbiotechnolosy p i e may b e c o n t a m i n a t e d w h e n the p r o d u c t s o f the first r o u n d reaction are transferred into the s e c o n d round, A n e w strategy, called 'drop-in, d r o p - o u t n e s t e d priming' [2], o v e r c o m e s this p r o b l e m b y designing an outer a n d an inner set o f p r i m e r s in s u c h a w a y that they will amplify u n d e r different thermal profiles. Erlich e t aL [2], discuss o n e a p p r o a c h to constructing t h e o u t e r p r i m e r s incorporating a GC-clamp, w h i c h e n s u r e s that the melting temperature o f the initial PCR p r o d u c t is high. In contrast, the inner p r i m e r s a r e m a d e relatively short o r AT-rich so that they c a n n o t b i n d to the t e m p l a t e at the high annealing t e m p e r a t u r e u s e d in the initial PCR cycling. A n u m b e r o f cycles are c o m p l e t e d b e f o r e switching f r o m the outer to the inner p r i m e r set b y d r o p p i n g the annealing temperature, thus allowing the i n n e r p r i m e r set to anneal. This is followed, after a further f e w cycles, b y lowering the denaturation t e m p e r a t u r e f u r t h e r to s t o p the initial PCR p r o d u c t from denaturing. T h e net effect o f these steps is nested PCR in a single tube. T h e c o m b i n a t i o n o f these n e w strategies will have implications in a n u m b e r o f areas involving the d e t e c t i o n o f m i n o r i t y cell populations.

Anti-contamination

measures

T h e sensitivity o f the PCR also results in o n e o f its main weaknesses, t h e p r o p e n s i t y f o r false-positive results d u e to contamination, particularly in high sensitivity applications involving the d e t e c t i o n o f single o r small numb e r s o f targets. Since the first anti-contamination protocols d e s c r i b e d in 1988 [31], various n e w strategies have b e e n suggested. A very p r o m i s i n g n e w a p p r o a c h , first described b y Longo e t al. [32 o.] involves the i n c o r p o r a t i o n o f dUTP instead o f T I P into all PCR p r o d u c t s and then treating all a s s e m b l e d PCR reaction m i x e s with the enzyme uracil DNA glycosytase, w h i c h selectively degrades any contaminating PCR p r o d u c t s containing dUTP. Ano t h e r n e w a p p r o a c h , d e s c r i b e d b y Cimino e t al. [33], incorporates i s o p s o r a l e n derivatives into PCR p r o d u c t s followed b y a p h o t o c h e m i c a l p r o c e s s w h i c h renders the PCR p r o d u c t i n c a p a b l e o f b e i n g amplified.

Conclusions

D e v e l o p m e n t s over the p a s t few years have p r o v i d e d us with a variety o f PCR-based m e t h o d s for characterizing h u m a n genes. As n o o n e m e t h o d is clearly s u p e r i o r to the others, researchers a n d w o r k e r s in molecular diagnostics will have to m a k e their c h o i c e o n the basis o f their particular requirements. O n e can e x p e c t to see a further diversification o f PCR-based m e t h o d s for genetic analysis, a n d hopefully t h e r e will b e a steady resolution o f sonde o f the potential p r o b l e m s associated with PCR, such as contamination.

Acknowledgements We thank the Wellcome Trust and the Foulkes Foundation Fellowship for support. We also thank JS wainscoat, KA Fleming, WZ Mehal and

P Patel for helpful discussion. Y-MD Lo is a Junior Research Fellow of Hertford College, Oxford, UK.

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 1.

SMKIILK,SCHARFS, FALOONAF, MULLISKB, HORN GT, ERIaCH HA, ARNt~IM N: Enzymatic Amplification of I~-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 1985, 230:1350-1354.

2.

ERLICHHA, GELFANDD, SNINSKYJJ: Recent Advances in the Polymerase Chain Reaction. Science 1991, 252:1643-1651.

3.

WmGrrrPA, W~NFORD-THoMASD: The Polymerase Chain Reaction: Miracle or Mirage? A Critical Review of its Uses and Limitations in Diagnosis and Research. J Patho/1990, 162:99-117.

4.

MCPHERSONMJ, QUIRKEP, TAYLORGR (EDS): PC.R:A Practical Approach [book]. Oxford: IRL Press, 1991.

5.

Wu DY, UGozzou L, PALBK, WALLACERB: Allele-Specific Enzymatic Amplification of ~-Globin Genomic DNA for Diagnosis of Sickle Cell A n e m i ~ Proc Natl Acad Sci U S A 1989, 86:2757-2760.

6.

NEWTONCR, GRAHAMA, HEPSTINSTALLLE, POWELLSJ, SUMMERS C, KAtSVa~KERN, Sirra JC, MARKHAMAF: Analysis of any Point Mutation in DN/L The Amplification Refractory Mutation System (ARMS). Nucleic Acids Res 1989, 17:2503-2516.

7.

LOY-MD, MEHALWZ, WORDSWORTHBP, CHAPMANRW, FLEMING KA, BELLJ1, WAINSCOATJS: HLA-Typing by Double ARMS. Lancet 1991, 338:65-66.

8.

CHEHA8FF, KAN YW: Detection of Specific DNA Sequence by Fluorescence Amplification: a Color Complementation Assay. Proc Natl Acad Sci U S A 1989, 86:9178-9182.

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CHASmERIA~ JS, GIBBSRA, RANIERJE, NGUYENPN, CASKEYCT: Deletion Screening of the Duchenne Muscular Dystrophy Locus Via Multiplex DNA Amplification. Nucleic Acids Res 1988, 16:11141-11156.

10.

l.L3 Y-MD, PATELP, NEWTONCR, MARKHAMAF, FLEMINGKA, WAINSEOAT JS: Direct Haplotype Determination by Double ARMS: Specificity, Sensitivity and Genetic Applications. Nu. cleic Acids Res 1991, 19:3561-3567. A detailed analysis of the application of double ARMStechniques for direct haplotype determination. This paper also discusses the genetic applications of double ARMSresulting from its enhanced specificityover single ARMS. •

11.

RUANOG, KIDD KK, STEPHENSJC: Haplotype of Multiple Polymorphisms Resolved by Enzymatic Amplification of Single DNA Molecules. Proc N a a Acad Sci U S A 1990, 87:6296-6300.

12.

Ia H, Cul X, ARmtEIMN: Direct Electrophoretic Detection of the Allelic State of Single DNA Molecules in Human Sperm by Using the Polymerase Chain Reaction. Proc Nail Acad Sci U S A 1990, 87:4580--4584.

13.

HAuhSSOSA, CHOMELJC, TESSON L, BAUDISM, KRUI-IJ, KAPLAN JC, KrlTas A: Modification of Enzymatically Amplified DNA for the Detection of Point Mutations. Nucleic Acids Res 1989, 17:3606.

14.

LINDEMANR, HU SP, VOLPATOF, TRENTRJ: Polymerase Chain Reaction (PCR) Mutagenesis Enabling Rapid Non-Radioactive Detection of Common 13-Thalassaemia Mutations in Mediterraneans. Br J Haematol 1991, 78:100-104.

Application of PCR to human gene detection Lo and Markham EIKENHG, ODLAND E, BOMAN H, SKJELKVALEL, ENGEBRETSEN LF, APOLDJ: Application of Natural and Amplification Created Restriction Sites for the Diagnosis of PKU Mutations. Nucleic Acids Res 1991, 19:1427-1430. A generally useful paper which discusses the theoretical basis for constructing A-RFLPfor genetic diagnosis.

hag Familial Hypercholesterolemia in Ashkenazi Jews. A m J H u m Genet 1991, 49:443--449.

15.



16.

PATELP, ~ Y - i n , BELLJI, WAINSCOATJS. Detection of Susceptibility Alleles to Insulin-Dependent Diabetes Mellitus at the DQB1 Locus by Artificial PCR-RFLP. Immunogenetics 1992, m press.

17.

TODDJA, BELLJI, MCDEVITI"HO: HLA-DQ Beta Gene Contributes to Susceptibility and Resistance to Insulin-Dependent Diabetes Mellitus. Nature 1987, 329:599-604.

18.

ORITAM, SUZUKIY, SEIKIYAT, HAYASH1K: Rapid and Sensitive Detection of Point Mutations and DNA Polymorphisms Using the Polymerase Chain Reaction. G e n o m ~ 1989, 5:874-879.

19.

LABRUNEP, MELLED, REY F, BERTHELONM, CAIILAUDC, REY J, MUNNICHA, LYONNETS: Single-Strand Conformation Polymorphism for Detection of Mutations and Base Substitutions in Phenylketonuria. Am J H u m Genet 1991, 48:1115-1120.

20.

AiNswoirraPJ, Sum LC, COULTER-MAcmXMB: Diagnostic Single Strand Conformation Polymorphism (SSCP): a Simplified Non-Radioisotopic Method as Applied to a Tay-Sachs B1 Variant. Nucleic Acids Res 1991, 19:405-406.

21.

YAP EPH, MCGEEJOD: Non-Isotopic SSCP Detection in PCR Products by Ethidium Bromide Staining. Trends Genet 1992, 8:489.

22.

23.

LO Y-MD, PATELP, MEHALWZ, FLEMINGKA, BELLJI, WAINSCOAT JS: Analysis of Complex Genetic Systems by ARMS-SSCP: Application to I-ILA Genotypin8 Nucleic Acids Res 1992, in press. NAGAMINECM, CHANK, LAUY-FC: A PCR Artifact: Generation of Heteroduplexes. Am J H u m Genet 1989, 45:337-339.

24.

Cm S-P, ENG B, KAN YW, CHUI DHK: A Rapid and Simple Electrophoretic Method for the Detection of Mutations Involving Small Insertion or Deletion: Application to ~-Thalas,~mia. H u m Genet 1991, 87:728-730.

25.

TmGGS-RAn',mBL, GRAVELP,A:Diagnostic Heteroduplexes: Simple Detection of Carriers of a 4-bp Insertion Mutation in Tay-Sachs Disease. Am J H u m Genet 1990, 46:183-184.

26.

MEINERV, LANDSBERGERD, BERKMANN, RESHEFA, SEGAL P, SEFTEL HC, VAN DER WESTHUYZEN DR, JEENAH MS, COETZEE GA, LEITERSDORFE: A Common Lithuanian Mutation Cans-

27. ..

SMITHB, SELBYP, SOUTHGATEJ, PrlTMAN K, BRADLEYC, BLAIR GE: Detection of Melanoma Cells in Peripheral Blood by Means of Reverse Transcriptase and Polymerase Chain Reaction. Lancet 1991, 338:1227-1229. An ingenious paper that describes the use of reverse transcriptase PCR m detecting circulating melanoma cells. A similar approach may be developed for other tumours and for the detection of other minority cell populations. 28. •

MYERSTW, GELFAND DH: Reverse Transcription and DNA Amplification by a Thermus Thermophilus DNA Polymerase. Biochemistry 1991, 30:7661-7666. Reports a new enzyme that allows efficient coupled reverse transcription and PCR amplification. New methodologies based on this enzyme should greatly simplify amplification from RNA sources. 29.

Lo Y-in, PATEL P, WAINSCOAT JS, SAMPIETRO M, GILLMErt MDG, FLEMINGKA: Prenatal Sex Determination by DNA Amplification from Maternal Peripheral Blood. Lancet 1989, ii:1363-1365.

30.

CAMASCHELLA C, ALEARANOA, GOTrARDI E, TRAVIM, PRIMIGNANI P, CAPPIO FC, SAGLIO G: Prenatal Diagnosis of Fetal Hemoglobin Lepore-Boston Disease on Maternal Peripheral Blood. B/oDd 1990, 75:2102-2106.

31.

Lo Y-MD, MEHALWZ, FLEMINGKA: False-Positive Results and the Polymerase Chain Reaction. Lancet 1988, //:679

32. **

LONGOMe, BERNINGERMS, HAR'IIEYJL: Use of Uracil DNA Glycosylase to Control Carry-Over Contamination in Polymerase Chain Reaction. Gene 1990, 93:125-128. Describes a promising approach for the control of carryover contamination. The sterilization phase takes place following the full assembly of PCR reactions and does not affect normal template DNA, PCR cycling following the sterilization process may therefore be carried out without re-opening the tubes. 33.

CIMINOGD, METCHETIE KC, TESSMANJW, HEARSTJE, ISAACS ST: Post-PCR Sterilization: a Method to Control Carryover Contamination for the Polymerase Chain Reaction. Nuc/eic Acids Res 1991, 19:99-107.

Y-MD Lo, Nuttleld Department of Pathology and Bacteriology, AF Markham, Department of Haematology, John RadcliffeHospital, Oxford OX3 9DU, UK.

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Application of PCR to human gene detection.

The advent of the polymerase chain reaction has stimulated the development of a number of rapid methods for characterizing human genes. In addition, t...
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