Molecular and Cellular Probes (1992) 6, 375-380
Tandem competitive polymerase chain reaction (TC-PCR) : a method for determining ratios of RNA and DNA templates Amarjit S . Virdi,'* Sanjeev Krishna 2 and Bryan C. Sykes' 2 'Collagen Genetics Group and MRC Molecular Haematology Unit, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK (Received 9 June 1992, Accepted 1 July 1992)
A sensitive and accurate method for determining the ratios of RNA and DNA templates by polymerase chain reaction (PCR) is presented . A common competitor containing tandemly arranged internal standards differing from the target template by the presence of different restriction enzyme sites is coamplified with the target templates under identical conditions . Products from each template and internal standard are identified by the band pattern after digestion with the restriction enzyme . As the amount of the common competitor is kept constant for all target templates, the ratio of PCR products from the templates reflects their ratio in the reaction mix before amplification . The method was used to study the relative abundance of mRNA for the pro-a, and pro-a ; chains of type I collagen and for estimating disturbances of normal ratio in the inherited bone disorder, osteogenesis imperfecta .
KEYWORDS: Competitive, quantitative, polymerase chain reaction, osteogenesis imperfecta .
INTRODUCTION nature of the reaction . In theory, the amplification of a template in PCR is an exponential process but it relies heavily on the efficiency of the reaction . In practice, factors (such as buffer, enzyme, temperature) which affect the efficiency even slightly can lead to large differences in the amount of the final product. Methods have been described recently which allow PCR to be used for quantitation of mRNA and DNA .', ' Termed competitive polymerase chain reaction (C-PCR), they include an internal standard in the amplification reaction which uses the same set of primers and is coamplified with the target template . The internal standard may vary from the target template by the presence or absence of a restriction
The ability to measure mRNA species accurately is important in many studies on the responses of biological systems to in vivo and in vitro stimuli. Convenfional methods employed to estimate relative or absolute amounts of mRNA include Northern and 'dot-blot' hybridization, nuclear protection mapping and in situ hybridization . In addition to providing only crude estimation of mRNA, these methods suffer from limited sensitivity and/or require a considerable amount of mRNA as a starting material . As is well known, the polymerase chain reaction (PCR)' exhibits high sensitivity and has been used widely for studying low abundance mRNA . The use of PCR as a direct means of quantitating mRNA, however, has proved to be difficult owing to the complex
' Author to whom correspondence should be addressed . 0890-8508/92/050375 + 06 $08 .00/0
375
© 1992 Academic Press Limited
A . S . Virdi et al.
376
enzyme site or the inclusion of a small piece of extra sequence .' The products generated by the internal standard can then be differentiated from those coming from the target template by digestion with the distinguishing restriction enzyme and separation on gel electrophoresis . The amount of products generated can be determined by densitometry or more accurately by the incorporation of radioactivity . As both templates are exposed to identical PCR conditions, the ratio of products at the end of the amplification is taken to equal their ratio at the start. When the amount of internal standard added to the reaction is known, the amount of the target template in the initial mixture can be calculated . These methods have been shown to be reliable in detecting and quantitating specific mRNA in samples of less than 0 . 1 ng of total RNA,' and also for mRNA from 200 cells .' Here we describe a method, based on C-PCR, which is intrinsically more accurate when the relative abundance of two (or more) templates needs to be estimated . The method differs from C-PCR in two main respects . First, it employs a common internal standard in which the two modified templates have been arranged in tandem fashion at 1 :1 ratio ; hence the term tandem competitive polymerase chain reaction (TC-PCR). In a modification which could equally be applied to straightforward competitive PCR, the restriction enzyme sites in the internal standards are not new sites but are generated by modifying the sequences for existing restriction enzyme sites . In this study TC-PCR was used to measure the relative abundance of mRNA for the pro-a, and pro-a 2 chains of type I collagen and for estimating disturbances of the normal ratios in fibroblasts grown from patients with the inherited bone disorder, osteogenesis imperfecta (01) .
I
MATERIALS AND METHODS Culture of human skin fibroblasts Skin biopsies were obtained from normal and affected individuals and cultured in vitro in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS) . Cultures were subdivided and expanded, and maintained in DMEM with 5% FCS . Before using the cultures, the medium was supplemented with ascorbic acid (50 pg ml - ') for 18 h .
RNA preparation and cDNA synthesis Fibroblasts at confluent monolayer stage were washed with sterile PBS and used directly for RNA extraction by the method of Chomczynski & Sacchi ." Up to 10 µg total RNA was used for cDNA synthesis by reverse transcription kit (Amersham) according to instructions .
Oligonucleotides Oligonucleotide primers spanning the 3' C-propeptide of COL1A1 and COL1A2 (Fig . 1) were synthesised on Applied Biosystem PCR Mate (Table 1) . 5.6 Primers for preparing modified templates were also prepared in similar fashion and are shown in Fig . 1 and Table 1 .
Polymerase chain reaction The standard reaction mixes contained PCR buffer (50 mm Tris-HCI pH 8-3,1-5 mm MgCl 2, 50 mm KCI, no gelatin or BSA), dNTPs (Pharmacia ; 250 µM final concentration each), Nonidet P-40 (1 :2000 final dilution), primers (50 pm), Taq DNA polymerase (Perkin-Elmer Cetus; 50 U ml -1 ) and template. Samples were over-
I
393 by
232 by
P6
P3 J
I
P2
P7 J 290 by
205 by P8
Map of COL1A1 and sequences see Table 1 . Fig. 1 .
COL1A2
COLIAI
COL 1A2 P4
genes showing primer sites and digest fragment sizes . For primer
377
Quantitative polymerase chain reaction Table 1 . Sequence and location of primer oligonucleotidess'b used for amplification and site-directed mutagenesis of COL1A1 and COL1A2 cDNAs . Underlined sequences indicate restriction sites for Bam HI incorporated in the competitor templates Primer - Locus
Sequence
Location
5' P1 P2 P3 P4 P5 P6 P7 P8
COL1A1 COL1A1 COL1A2 COL1A2 COL1A1 COL1A1 COL1A2 COL1A2
ATG TTG TGC TTG CAG CGT GCT GTC
3' TCT GTC TGG CAC ACC CGA GAC CAC
GGT ATG CCA AAT AGG AGC CAT AAA
laid with light paraffin oil and amplified in an Autogene cycling water bath (Grant Instruments, Cambridge, UK) . The amplification profile involved pretreatment at 93 ° C for 3 min, followed by cycling (up to 30 times) of primer annealing and extension at 62 ° C for 2 min and denaturation at 93 ° C for 30 s. Finally, the mixture was subjected to post-cycling treatment of primer extension at 62 ° C for 10 min . For estimating the PCR products, radiolabelled [a 32 P]dCTP was incorporated into the reaction mix (20 µCi ml - ' [6 . 6 nMl).
Construction of the internal standards Internal standards were prepared according to the method of Higuchi et al .' Briefly, primers containing the modified sequence (Table 1) were used together with the flanking primers in PCR to give products which contained a Barn HI site instead of an Eco RI site (Fig . 1) .
Cloning PCR products were ligated into Sma I cut pUC13 vector and used for transforming DH5a competent cells (Boehringer Mannheim) according to suppliers' instructions. Plasmid preparations were carried out according to standard methods .' Plasmid TC-1 is the common internal standard containing COL1A1 and COL1A2 PCR products with Barn HI sites . To construct TC-1, two separate clones with Barn HI sites were generated as described above . These were digested with Eco RI and ligated in the fashion shown in Fig. 2(a) . To select for fragments containing the two sequences in tandem orientation, primers P1 and P4 were used to amplify across the full, length (Fig . 2b) .
TCG TTC ACT GCT GAT CGG GGA GGA
GCG GGT ATG CTG CCG ATC TCC TCC
AGA TGG CCT ATC GCT CCT TTT ATG
GC TC CT TCG GGT GTG GTC
ACG CTG GAC AGC
4078-4097 4703-4684 3911-3930 4406-4389 4459-4482 4482-4459 4189-4212 4212-4189
This product was then ligated into Sma I cut pUC13 to give the final plasmid TC-1 and cloned (Fig . 2c) .
Quantitative analysis by TC-PCR In order to determine the ratio of COL1A1 and COL1A2 in a solution, each template was titrated against an identical doubling dilution series of TC-1 . Primer set P1 and P2 were used for COL1A1/TC-1 and P3 and P4 for COL1A2/TC-1 . PCR conditions were as described above and [a12P]-dCTP was used in the reaction mix. After amplification, the products were digested with Eco RI (or Barn HI) and separated on 1 . 5% (w/v) agarose gel . Bands were excised and counted by the Cerenkov method . Counts in each band were represented as percentage of total counts recovered for the track .
RESULTS AND DISCUSSION Although PCR has proved a versatile tool for studying very small amounts of specific mRNA/DNA templates, its deployment for direct quantitation of these molecules has resulted in problems . Gilliland et al .' reported up to six-fold variation in the amplified products within the same dilution of template even when master reagent mixes were used throughout the protocol . We have observed similar variations during estimation of relative amounts of two templates under identical conditions (data not shown) . With the introduction of C-PCR it has been possible to use the reaction for quantitating target templates with much better reliability and accuracy" and the method has been applied successfully to a number of templates including low abundance mRNA . Where accurate determination of relative
378
A . S . Virdi et al. Ice Z"
Amp r
Ice Z"
Ice Z"
Amp r
lac Z"
(a )
( b ) r P4
(c )
Fig. 2. Diagrammatical representation of the common competitor TC-1 . (a) Tandem ligation of COL1A1 and COL1A2 clones containing mutant (Bam HI) sites; (b) selection of tandem orientation by using primers P1 and P4; (c) subcloning of amplified TC-1 into pUC13 by Sma I cut blunt ended ligation .
abundance of two or more mRNA species is required, it is necessary to set up independent reactions for all appropriate mRNA species . The accuracy of this method relies heavily on the precise determination of the internal competitor concentrations in the initial reaction mix. Any errors at this point will lead to an inaccurate estimate of the relative abundance of the targets . In our case, we were interested in estimating the relative abundance of mRNA from collagen type I genes COL1A1 and COL1A2 (Fig . 1) in normals and individuals suffering from mild forms of osteogenesis imperfecta (Sillence Type I) .' In some cases the expression of one of the alleles at the affected locus is reduced and we wanted to design a rapid method for detecting when this occurred . Our modification of the competitive PCR concept involves preparing internal competitors for all relevant templates and ligating them to each other in a tandem fashion in 1 :1 ratio . This ensures that in subsequent steps the internal standards for all target templates are always present in the same ratio. It follows that, when the same amount of tandem internal standard is used in separate amplifications of the two, or more, different targets, their relative abundance is exactly equal to the ratio of the proportion each contributes to the amplified products in the separate reactions . The crucial advantage of the modification is that it is not necessary to know the concentrations of the internal standards .
An example of such a common competitor is shown in Fig . 2(c) where two modified templates for COL1A1 and COL1A2 are ligated and cloned into pUC13 Sma I cut vector (TC-1) . To estimate the ratio of COL1A1 to COL1A2 mRNA in cultured dermal fibroblasts, total RNA from the cells was prepared and converted to cDNA by reverse transcription (Amersham) . Both cDNAs were co-amplified with an identical dilution series (1 :2) of TC-1 (Fig . 3a) using appropriate primer sets (Table 1). The amplification products were digested with Eco RI and separated on 1 . 5% (w/v) agarose (Fig. 3b) . Primer set P1 and P2 for COL1A1 gives a product of 625 by which upon digestion with Eco RI results in two fragments of 393 by and 232 bp . Since the cDNA template contains an Eco RI site, the enzyme digests the products originating from this template only but not from the internal standard . For COL1A2 the primer set P3 and P4 give a 495 by product which results in 290 by and 205 by fragments on cleavage at the Eco RI site in this target . Conversely these products may be digested with Bam HI to show the digested fragments originating from the engineered sites in the internal standard . Any formation of heteroduplexes between the products of target and standard templates will be seen as fragments on the gel that are resistant to simultaneous or sequential digestion by both enzymes . For a given dilution series, the amount of product from TC1 decreases with dilution and there is a correspond-
Quantitative polymerase chain reaction
379
(a ) Competitor template TC-I
Competitor template TC-I
I Target template COL IAI
I
I Target template COL IA2
(b)
by
by 625
495 393 290 205
232 (a)
Dilution step
Dilution step
Titration experiment in which COL1A1 and COL1A2 templates were titrated against an identical series of the common competitor TC-1 (a) . PCR products were digested with Eco RI and separated on 1-5% agarose gel (b). Bands were cut, counted, represented as a percentage of total counts in the track and plotted against dilution step of TC-1 (c). See text for detail . 0, Undigested COL1A1 products (%); ∎, digested COL1A1 products (%) ; 0, undigested COL1A2 products (%) ; •, digested COL1A2 products (%) . Fig . 3 .
ing increase in the products generated from the cDNA template. The pattern on the gel results from the competitive nature of the reaction in which the template bias is initially in favour of TC-1 but swings towards cDNA when the TC-1 concentration falls below that of the cDNA (Fig . 3b) . A point where the products amplified from either template are present in equal amounts is the dilution point where both templates are present at equal levels in the initial reaction mix. It is quite easy to see this point on the gel but for more accurate determination, bands were cut out and the products estimated by counting [a 32 P]-dCTP incorporation . To avoid possible errors during loading of the gel, counts in each band were represented as a percentage of the total counts in all three bands in the track . These were then reported as percentage of total for digested and undigested products and plotted against dilution step (Fig . 3c) . The cross-over point of curves for digested and
undigested products is taken as the dilution at which both cDNA and internal standard template concentrations in the pre-amplification reaction mix are the same . When cDNA from COL1A1 and COL1A2 are titrated against an identical dilution series of TC-1, the cross-over points for both reactions should be the same if the two cDNAs are present in equal amounts in a sample . A shift in this point for one cDNA template relative to the other towards the left of the graph indicates that this template is present at a higher level and the relative abundance can be calculated by the degree of shift . Similarly, a shift towards the right of the graph suggests that the template is present in relatively lower amounts than the other template . Figure 4 shows the pedigree of a family affected with mild 01 (type I) . Linkage analysis had previously shown that the disorder in this family segregated with COL1A1 . 10 Ratios of COL1A1 and
380
A . S . Virdi et al.
I
REFERENCES
0
* o * o b
1--o
Fig . 4 . Pedigree of a family affected with mild osteogenesis imperfecta (type I) . Individuals tested are indicated with arrows. COL1A2 mRNA were estimated in one normal and one affected invididuals in this family using TC-PCR (Fig . 5) . In the normal individual the ratio of COL1A1 to COL1A2 was found to be 1 . 04:1 (Fig . 5a) whereas in affected individuals the ratio was 0 . 46 :1 (Fig. 5b) . The most likely explanation is that the patient had one normal and one non-expressed allele which, in turn, gave rise to the phenotype . TC-PCR is a generally applicable method allowing direct comparisons of mRNA abundances . It does not depend on equal amplification rates, nor on knowledge of the internal standard concentrations, nor on avoidance of heteroduplex formation . Since the method compares cDNA concentrations, extrapolation to mRNA abundances does depend on there being equivalent reverse transcription rates during cDNA synthesis . Though our construct contained only two competitor templates, there seems to be no reason why a large number of tandemly arranged engineered internal standards could not be incorporated into the same competitor clone . Neither is there any reason why the same general method could not be used for genomic DNA comparisons, for example, of gene dosage estimates where current methods are subject to many of the limitations inherent in estimating mRNA abundances .
1 . Saiki, R ., Scharf, S ., Faloona, F ., Mullis, K . B ., Horn, G. T ., Erlich, H . A . & Arnheim, N . (1985) . Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia . Science 230,1350-4 . 2 . Wang, A. M ., Doyle, M . V. & Mark, D . F . (1989) . Quantification of mRNA by the polymerase chain reaction [published erratum appears in Proc Natl Acad Sci USA 1990, 87 :28651 Proceedings of the National Academy of Sciences, USA 86, 9717-21 . 3 . Gilliland, G ., Perrin, S ., Blanchard, K . & Bunn, H . F . (1990). Analysis of cytokine mRNA and DNA : detection and quantitation by competitive polymerase chain reaction . Proceedings of the National Academy of Sciences, USA 87,2725-9 . 4. Chomczynski, P . & Sacchi, N . (1987) . Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction . Analytical Biochemistry 162, 156-9 . 5 . Bernard, M . P ., Chu, M .-L ., Myers, J . C ., Ramirez, F ., Eikenberry, E. F . & Prockop, D . J. (1983). Nucleotide sequences of complementary deoxyribonucleic acids for the pro alpha 1 chain of human type I procollagen . Statistical evaluation of structures that are conserved during evolution . Biochemistry 22, 5213-23 . 6. de Wet, W., Bernard, M ., Benson-Chanda, V ., Chu, M .L ., Dickson, L ., Weil, D . & Ramirez, F . (1987) . Organization of the human pro-alpha 2(I) collagen gene . Journal of Biological Chemistry 262, 16032-6. 7. Higuchi, R ., Krummel, B . & Saiki, R . K . (1988) . A general method of in vitro preparation and specific mutagenesis of DNA fragments : study of protein and DNA interactions. Nucleic Acids Research 16, 7351-67. 8. Birnboim, H . C . & Doty, J . (1979) . A rapid alkaline extraction procedure for screening recombinant plasmid DNA . Nucleic Acids Research 7, 1513-23 . 9. Sillence, D . 0 ., Senn, A . & Danks, D . M . (1979) . Genetic heterogeneity in osteogenesis imperfecta . Journal of Medical Genetics 16, 101-16. 10 . Sykes, B ., Ogilvie, D ., Wordsworth, P., Anderson, J . & Jones, N . (1986) . Osteogenesis imperfecta is linked to both type I collagen structural genes . Lancet ii, 69-72 .
0 2
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7
8
9
10
4
5
6
7
8
9
10
Dilution step Fig. 5. Ratio of COL1A1 and COL1A2 mRNA in normal and affected individuals of a family affected with mild osteogenesis imperfecta . (a) Normal female, and (b) affected female . Key as for Fig. 3 .
Tandem competitive polymerase chain reaction (TC-PCR) : a method for determining ratios of RNA and DNA templates Amarjit S . Virdi,'* Sanjeev Krishna 2 and Bryan C. Sykes' 2 'Collagen Genetics Group and MRC Molecular Haematology Unit, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK (Received 9 June 1992, Accepted 1 July 1992)
A sensitive and accurate method for determining the ratios of RNA and DNA templates by polymerase chain reaction (PCR) is presented . A common competitor containing tandemly arranged internal standards differing from the target template by the presence of different restriction enzyme sites is coamplified with the target templates under identical conditions . Products from each template and internal standard are identified by the band pattern after digestion with the restriction enzyme . As the amount of the common competitor is kept constant for all target templates, the ratio of PCR products from the templates reflects their ratio in the reaction mix before amplification . The method was used to study the relative abundance of mRNA for the pro-a, and pro-a ; chains of type I collagen and for estimating disturbances of normal ratio in the inherited bone disorder, osteogenesis imperfecta .
KEYWORDS: Competitive, quantitative, polymerase chain reaction, osteogenesis imperfecta .
INTRODUCTION nature of the reaction . In theory, the amplification of a template in PCR is an exponential process but it relies heavily on the efficiency of the reaction . In practice, factors (such as buffer, enzyme, temperature) which affect the efficiency even slightly can lead to large differences in the amount of the final product. Methods have been described recently which allow PCR to be used for quantitation of mRNA and DNA .', ' Termed competitive polymerase chain reaction (C-PCR), they include an internal standard in the amplification reaction which uses the same set of primers and is coamplified with the target template . The internal standard may vary from the target template by the presence or absence of a restriction
The ability to measure mRNA species accurately is important in many studies on the responses of biological systems to in vivo and in vitro stimuli. Convenfional methods employed to estimate relative or absolute amounts of mRNA include Northern and 'dot-blot' hybridization, nuclear protection mapping and in situ hybridization . In addition to providing only crude estimation of mRNA, these methods suffer from limited sensitivity and/or require a considerable amount of mRNA as a starting material . As is well known, the polymerase chain reaction (PCR)' exhibits high sensitivity and has been used widely for studying low abundance mRNA . The use of PCR as a direct means of quantitating mRNA, however, has proved to be difficult owing to the complex
' Author to whom correspondence should be addressed . 0890-8508/92/050375 + 06 $08 .00/0
375
© 1992 Academic Press Limited
A . S . Virdi et al.
376
enzyme site or the inclusion of a small piece of extra sequence .' The products generated by the internal standard can then be differentiated from those coming from the target template by digestion with the distinguishing restriction enzyme and separation on gel electrophoresis . The amount of products generated can be determined by densitometry or more accurately by the incorporation of radioactivity . As both templates are exposed to identical PCR conditions, the ratio of products at the end of the amplification is taken to equal their ratio at the start. When the amount of internal standard added to the reaction is known, the amount of the target template in the initial mixture can be calculated . These methods have been shown to be reliable in detecting and quantitating specific mRNA in samples of less than 0 . 1 ng of total RNA,' and also for mRNA from 200 cells .' Here we describe a method, based on C-PCR, which is intrinsically more accurate when the relative abundance of two (or more) templates needs to be estimated . The method differs from C-PCR in two main respects . First, it employs a common internal standard in which the two modified templates have been arranged in tandem fashion at 1 :1 ratio ; hence the term tandem competitive polymerase chain reaction (TC-PCR). In a modification which could equally be applied to straightforward competitive PCR, the restriction enzyme sites in the internal standards are not new sites but are generated by modifying the sequences for existing restriction enzyme sites . In this study TC-PCR was used to measure the relative abundance of mRNA for the pro-a, and pro-a 2 chains of type I collagen and for estimating disturbances of the normal ratios in fibroblasts grown from patients with the inherited bone disorder, osteogenesis imperfecta (01) .
I
MATERIALS AND METHODS Culture of human skin fibroblasts Skin biopsies were obtained from normal and affected individuals and cultured in vitro in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS) . Cultures were subdivided and expanded, and maintained in DMEM with 5% FCS . Before using the cultures, the medium was supplemented with ascorbic acid (50 pg ml - ') for 18 h .
RNA preparation and cDNA synthesis Fibroblasts at confluent monolayer stage were washed with sterile PBS and used directly for RNA extraction by the method of Chomczynski & Sacchi ." Up to 10 µg total RNA was used for cDNA synthesis by reverse transcription kit (Amersham) according to instructions .
Oligonucleotides Oligonucleotide primers spanning the 3' C-propeptide of COL1A1 and COL1A2 (Fig . 1) were synthesised on Applied Biosystem PCR Mate (Table 1) . 5.6 Primers for preparing modified templates were also prepared in similar fashion and are shown in Fig . 1 and Table 1 .
Polymerase chain reaction The standard reaction mixes contained PCR buffer (50 mm Tris-HCI pH 8-3,1-5 mm MgCl 2, 50 mm KCI, no gelatin or BSA), dNTPs (Pharmacia ; 250 µM final concentration each), Nonidet P-40 (1 :2000 final dilution), primers (50 pm), Taq DNA polymerase (Perkin-Elmer Cetus; 50 U ml -1 ) and template. Samples were over-
I
393 by
232 by
P6
P3 J
I
P2
P7 J 290 by
205 by P8
Map of COL1A1 and sequences see Table 1 . Fig. 1 .
COL1A2
COLIAI
COL 1A2 P4
genes showing primer sites and digest fragment sizes . For primer
377
Quantitative polymerase chain reaction Table 1 . Sequence and location of primer oligonucleotidess'b used for amplification and site-directed mutagenesis of COL1A1 and COL1A2 cDNAs . Underlined sequences indicate restriction sites for Bam HI incorporated in the competitor templates Primer - Locus
Sequence
Location
5' P1 P2 P3 P4 P5 P6 P7 P8
COL1A1 COL1A1 COL1A2 COL1A2 COL1A1 COL1A1 COL1A2 COL1A2
ATG TTG TGC TTG CAG CGT GCT GTC
3' TCT GTC TGG CAC ACC CGA GAC CAC
GGT ATG CCA AAT AGG AGC CAT AAA
laid with light paraffin oil and amplified in an Autogene cycling water bath (Grant Instruments, Cambridge, UK) . The amplification profile involved pretreatment at 93 ° C for 3 min, followed by cycling (up to 30 times) of primer annealing and extension at 62 ° C for 2 min and denaturation at 93 ° C for 30 s. Finally, the mixture was subjected to post-cycling treatment of primer extension at 62 ° C for 10 min . For estimating the PCR products, radiolabelled [a 32 P]dCTP was incorporated into the reaction mix (20 µCi ml - ' [6 . 6 nMl).
Construction of the internal standards Internal standards were prepared according to the method of Higuchi et al .' Briefly, primers containing the modified sequence (Table 1) were used together with the flanking primers in PCR to give products which contained a Barn HI site instead of an Eco RI site (Fig . 1) .
Cloning PCR products were ligated into Sma I cut pUC13 vector and used for transforming DH5a competent cells (Boehringer Mannheim) according to suppliers' instructions. Plasmid preparations were carried out according to standard methods .' Plasmid TC-1 is the common internal standard containing COL1A1 and COL1A2 PCR products with Barn HI sites . To construct TC-1, two separate clones with Barn HI sites were generated as described above . These were digested with Eco RI and ligated in the fashion shown in Fig. 2(a) . To select for fragments containing the two sequences in tandem orientation, primers P1 and P4 were used to amplify across the full, length (Fig . 2b) .
TCG TTC ACT GCT GAT CGG GGA GGA
GCG GGT ATG CTG CCG ATC TCC TCC
AGA TGG CCT ATC GCT CCT TTT ATG
GC TC CT TCG GGT GTG GTC
ACG CTG GAC AGC
4078-4097 4703-4684 3911-3930 4406-4389 4459-4482 4482-4459 4189-4212 4212-4189
This product was then ligated into Sma I cut pUC13 to give the final plasmid TC-1 and cloned (Fig . 2c) .
Quantitative analysis by TC-PCR In order to determine the ratio of COL1A1 and COL1A2 in a solution, each template was titrated against an identical doubling dilution series of TC-1 . Primer set P1 and P2 were used for COL1A1/TC-1 and P3 and P4 for COL1A2/TC-1 . PCR conditions were as described above and [a12P]-dCTP was used in the reaction mix. After amplification, the products were digested with Eco RI (or Barn HI) and separated on 1 . 5% (w/v) agarose gel . Bands were excised and counted by the Cerenkov method . Counts in each band were represented as percentage of total counts recovered for the track .
RESULTS AND DISCUSSION Although PCR has proved a versatile tool for studying very small amounts of specific mRNA/DNA templates, its deployment for direct quantitation of these molecules has resulted in problems . Gilliland et al .' reported up to six-fold variation in the amplified products within the same dilution of template even when master reagent mixes were used throughout the protocol . We have observed similar variations during estimation of relative amounts of two templates under identical conditions (data not shown) . With the introduction of C-PCR it has been possible to use the reaction for quantitating target templates with much better reliability and accuracy" and the method has been applied successfully to a number of templates including low abundance mRNA . Where accurate determination of relative
378
A . S . Virdi et al. Ice Z"
Amp r
Ice Z"
Ice Z"
Amp r
lac Z"
(a )
( b ) r P4
(c )
Fig. 2. Diagrammatical representation of the common competitor TC-1 . (a) Tandem ligation of COL1A1 and COL1A2 clones containing mutant (Bam HI) sites; (b) selection of tandem orientation by using primers P1 and P4; (c) subcloning of amplified TC-1 into pUC13 by Sma I cut blunt ended ligation .
abundance of two or more mRNA species is required, it is necessary to set up independent reactions for all appropriate mRNA species . The accuracy of this method relies heavily on the precise determination of the internal competitor concentrations in the initial reaction mix. Any errors at this point will lead to an inaccurate estimate of the relative abundance of the targets . In our case, we were interested in estimating the relative abundance of mRNA from collagen type I genes COL1A1 and COL1A2 (Fig . 1) in normals and individuals suffering from mild forms of osteogenesis imperfecta (Sillence Type I) .' In some cases the expression of one of the alleles at the affected locus is reduced and we wanted to design a rapid method for detecting when this occurred . Our modification of the competitive PCR concept involves preparing internal competitors for all relevant templates and ligating them to each other in a tandem fashion in 1 :1 ratio . This ensures that in subsequent steps the internal standards for all target templates are always present in the same ratio. It follows that, when the same amount of tandem internal standard is used in separate amplifications of the two, or more, different targets, their relative abundance is exactly equal to the ratio of the proportion each contributes to the amplified products in the separate reactions . The crucial advantage of the modification is that it is not necessary to know the concentrations of the internal standards .
An example of such a common competitor is shown in Fig . 2(c) where two modified templates for COL1A1 and COL1A2 are ligated and cloned into pUC13 Sma I cut vector (TC-1) . To estimate the ratio of COL1A1 to COL1A2 mRNA in cultured dermal fibroblasts, total RNA from the cells was prepared and converted to cDNA by reverse transcription (Amersham) . Both cDNAs were co-amplified with an identical dilution series (1 :2) of TC-1 (Fig . 3a) using appropriate primer sets (Table 1). The amplification products were digested with Eco RI and separated on 1 . 5% (w/v) agarose (Fig. 3b) . Primer set P1 and P2 for COL1A1 gives a product of 625 by which upon digestion with Eco RI results in two fragments of 393 by and 232 bp . Since the cDNA template contains an Eco RI site, the enzyme digests the products originating from this template only but not from the internal standard . For COL1A2 the primer set P3 and P4 give a 495 by product which results in 290 by and 205 by fragments on cleavage at the Eco RI site in this target . Conversely these products may be digested with Bam HI to show the digested fragments originating from the engineered sites in the internal standard . Any formation of heteroduplexes between the products of target and standard templates will be seen as fragments on the gel that are resistant to simultaneous or sequential digestion by both enzymes . For a given dilution series, the amount of product from TC1 decreases with dilution and there is a correspond-
Quantitative polymerase chain reaction
379
(a ) Competitor template TC-I
Competitor template TC-I
I Target template COL IAI
I
I Target template COL IA2
(b)
by
by 625
495 393 290 205
232 (a)
Dilution step
Dilution step
Titration experiment in which COL1A1 and COL1A2 templates were titrated against an identical series of the common competitor TC-1 (a) . PCR products were digested with Eco RI and separated on 1-5% agarose gel (b). Bands were cut, counted, represented as a percentage of total counts in the track and plotted against dilution step of TC-1 (c). See text for detail . 0, Undigested COL1A1 products (%); ∎, digested COL1A1 products (%) ; 0, undigested COL1A2 products (%) ; •, digested COL1A2 products (%) . Fig . 3 .
ing increase in the products generated from the cDNA template. The pattern on the gel results from the competitive nature of the reaction in which the template bias is initially in favour of TC-1 but swings towards cDNA when the TC-1 concentration falls below that of the cDNA (Fig . 3b) . A point where the products amplified from either template are present in equal amounts is the dilution point where both templates are present at equal levels in the initial reaction mix. It is quite easy to see this point on the gel but for more accurate determination, bands were cut out and the products estimated by counting [a 32 P]-dCTP incorporation . To avoid possible errors during loading of the gel, counts in each band were represented as a percentage of the total counts in all three bands in the track . These were then reported as percentage of total for digested and undigested products and plotted against dilution step (Fig . 3c) . The cross-over point of curves for digested and
undigested products is taken as the dilution at which both cDNA and internal standard template concentrations in the pre-amplification reaction mix are the same . When cDNA from COL1A1 and COL1A2 are titrated against an identical dilution series of TC-1, the cross-over points for both reactions should be the same if the two cDNAs are present in equal amounts in a sample . A shift in this point for one cDNA template relative to the other towards the left of the graph indicates that this template is present at a higher level and the relative abundance can be calculated by the degree of shift . Similarly, a shift towards the right of the graph suggests that the template is present in relatively lower amounts than the other template . Figure 4 shows the pedigree of a family affected with mild 01 (type I) . Linkage analysis had previously shown that the disorder in this family segregated with COL1A1 . 10 Ratios of COL1A1 and
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REFERENCES
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Fig . 4 . Pedigree of a family affected with mild osteogenesis imperfecta (type I) . Individuals tested are indicated with arrows. COL1A2 mRNA were estimated in one normal and one affected invididuals in this family using TC-PCR (Fig . 5) . In the normal individual the ratio of COL1A1 to COL1A2 was found to be 1 . 04:1 (Fig . 5a) whereas in affected individuals the ratio was 0 . 46 :1 (Fig. 5b) . The most likely explanation is that the patient had one normal and one non-expressed allele which, in turn, gave rise to the phenotype . TC-PCR is a generally applicable method allowing direct comparisons of mRNA abundances . It does not depend on equal amplification rates, nor on knowledge of the internal standard concentrations, nor on avoidance of heteroduplex formation . Since the method compares cDNA concentrations, extrapolation to mRNA abundances does depend on there being equivalent reverse transcription rates during cDNA synthesis . Though our construct contained only two competitor templates, there seems to be no reason why a large number of tandemly arranged engineered internal standards could not be incorporated into the same competitor clone . Neither is there any reason why the same general method could not be used for genomic DNA comparisons, for example, of gene dosage estimates where current methods are subject to many of the limitations inherent in estimating mRNA abundances .
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Dilution step Fig. 5. Ratio of COL1A1 and COL1A2 mRNA in normal and affected individuals of a family affected with mild osteogenesis imperfecta . (a) Normal female, and (b) affected female . Key as for Fig. 3 .
