Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder
Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binderIrina Afonina*, Maris Zivarts, Igor Kutyavin, Eugeny Lukhtanov, Howard Gamper and Rich B. Meyer
Epoch Pharmaceuticals Inc., 1725 220th Street SE, #104 Bothell, WA 98021, USA
Received February 25, 1997;Revised and Accepted May 19, 1997
ABSTRACT
The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole- 7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3 group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.
INTRODUCTION
PCR has become an exceptionally powerful tool in molecular biology, but certain factors limit its versatility. Under the highly stringent conditions optimal for activity of a thermophilic polymerase, for instance, short primers cannot be used. Primers of ~20 nucleotides in length (1 ) are normally used, since only ODNs of that length will form stable enough hybrids. There are important new techniques, such as gene hunting and differential display, for which a short primer is more appropriate, if not essential. In gene hunting, a family of amplified transcripts shares a short degenerate sequence that specifies a conserved peptide motif, and this priming sequence is necessarily limited in length (2 ). In differential display, complete representation of a transcript pool is sought, and this would be best achieved priming with 6mers. The impracticality of using such short primers necessitates the use of longer degenerate ODNs (3 ), which may not provide an accurate representation of the complexity of an mRNA population. Mis-priming can generate non-specific bands, and inefficient hybridization of the primer can lead to an under representation of some transcripts (4 ). Viral diagnostic applications have limitations because amplification of a common sequence from multiple strains can be complicated by the presence of genomic variability (5 ). Short ODNs would circumvent this problem by shortening the conserved sequences from which to prime.
By chemically modifying the primers to improve hybrid stability while still retaining good priming ability, it should be possible to shorten their length. Some such modifications have been shown to enhance hybrid stability but do not have a 3'-OH for extension, including N3' -> P5' phosphoramidates (6 ) and peptide (7 ) or guanidine (8 ) linkages. Other hybrid-stabilizing modifications that have not been shown to support primer extension are 2'-modified sugars (9 ,10 ), conjugated intercalating agents (11 ) and substituted bases such as 2-aminoadenine (12 ) or C5 propynyl pyrimidines (13 ).
We report here that a modification which greatly improves hybrid stability also allows the ODN to serve as a PCR primer. CDPI3 [the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole- 7-carboxylate, or CDPI] is a synthetic non-reactive derivative of a subunit of the antitumor antibiotic CC-1065 (14 ). This oligopeptide is a DNA minor groove binder (MGB), with a very high affinity for the minor groove of A-T-rich double-stranded DNA. We have previously reported that, when compared to unmodified ODNs of the same length, CDPI3-ODN conjugates form unusually stable and specific hybrids with complementary single-stranded DNA (15 ,16 ). We now report that conjugates of short ODNs with CDPI3 make effective primers for PCR. As a result, the MGB-ODN conjugates improve the yield and accuracy of priming. ODNs as short as 8mers and G-C-rich 6mers are able to specifically prime the amplification reaction when conjugated to an MGB.
MATERIALS AND METHODS
Oligonucleotides
DNA synthesis was performed on an Applied Biosystems Model 394 DNA synthesizer using the 1 [mu]mol coupling program supplied by the manufacturer. CDPI3 was postsynthetically conjugated to the 5'-end of ODNs as described by Lukhtanov et al. (15 ). ODNs were purified by HPLC on a reverse-phase column eluted by an acetonitrile gradient (usually 0-45%) in 100 mM triethylamine acetate (pH 7.5) buffer. Purity of unmodified ODNs was evaluated by electrophoresis on an 8% polyacrylamide-8 M urea gel with subsequent visualization by silver staining (Daiichi). Purity of the ODN-CDPI3 conjugates was verified by analytical HPLC as described above. All ODN preparations had >95% purity.
. Melting temperatures and properties of primers in this study
ODN
Tma (oC)
Length
%GC
Sequenceb
Locationc
1
45
16
37.5
5'-ATAAAACAGAGGTGAG-3'
4937-4922d
2
39
12
33.3
5'-ATAAAACAGAGG-3'
4937-4926d
2-C
56
12
33.3
5'-MGB-ATAAAACAGAGG-3'
4937-4926d
3
24
10
20
5'-ATAAAACAGA-3'
4937-4928d
3-C
46
10
20
5'-MGB-ATAAAACAGA-3'
4937-4928d
4
50
16
43.8
5'-TAATAACGTTCGGGCA-3'
4630-4645
4-C
66
16
43.8
5'-MGB-TAATAACGTTCGGGCA-3'
4630-4645
5
16
6
33.3
5'-ATAACG-3'
4632-4637
5-C
36
6
33.3
5'-MGB-ATAACG-3'
4632-4637
6-C
57
12
20
5'-MGB-TAATAACGTTCG-3'
4630-4641
7-C
49
10
20
5'-MGB-TAATAACGTT-3'
4630-4639
8
25
8
62.5
5'-CGGGCAAA-3'
4640-4647
8-C
33
8
62.5
5'-MGB-CGGGCAAA-3'
4640-4647
9
47
16
43.8
5'-CGGGCAAAGGATTTAA-3'
4640-4655
9-C
53
16
43.8
5'-MGB-CGGGCAAAGGATTTAA-3'
4640-4655
10
<17
6
50
5'-GGCAAA-3'
4642-4647
10-C
<17
6
50
5'-MGB-GGCAAA-3'
4642-4647
11
23
8
62.5
5'-CGGCTCTA-3'
4720-4727
11-C
37
8
62.5
5'-MGB-CGGCTCTA-3'
4720-4727
12
43
16
44
5'-CGGCTCTAATCTATTA-3'
4720-4735
12-C
52
16
44
5'-MGB-CGGCTCTAATCTATTA-3'
4720-4735
13
34
16
18.75
5'-TATTTTAGATAACCTT-3'
4756-4771
13-C
56
16
18.75
5'-MGB-TATTTTAGATAACCTT-3'
4756-4771
aMelting temperature of a duplex made with complementary ODN.
bMGB, CDPI3 tethered to 5'-phosphate (Fig. 1).
cLocation on M13 MP19 plasmid DNA.
dComplementary to the indicated sequence.
Thermal denaturation studies
Hybrids formed between MGB-tailed conjugates or unmodified ODNs and their complements were melted at a rate of 0.5oC/min in 140 mM KCl, 10 mM MgCl2 and 20 mM HEPES-HCl (pH 7.2) on a Lambda 2S (Perkin Elmer) spectrophotometer with a PTP-6 automatic multicell temperature programmer. Each ODN (2 [mu]M) was mixed with sufficient complement to give a 1:1 ratio. Prior to melting, samples were denatured at 100oC and then cooled to the starting temperature over a 10 min period. The melting temperatures (Tm) of the hybrids were determined from the derivative maxima and collected in Table 1 .
PCR reactions
All PCR reactions were performed on Perkin-Elmer Cetus DNA Thermocycler and included: PCR buffer (Promega) with no magnesium, 1.6 mM MgCl2, 50[mu]M dNTP, 50 nM each primer, 0.2 [mu]g M13mp19 DNA and 1-2 U Taq DNA polymerase (Promega). Final volume for each reaction was 50 [mu]l. The standard PCR profile was as follows: 3 min at 94oC, 30 cycles of 1 min at 94oC, 1.5 min at annealing temperature and 30 s at 72oC, finally followed by 5 min at 72oC and a 4oC soak. For the 8mers and 6mers, PCR was performed in the touch-down manner (17 ) with a starting annealing temperature of 55oC for 8mer primers and 50oC for 6mers. Each subsequent cycle had an annealing temperature 1oC lower until 41oC (for 8mers) or 37oC (for 6mers) was reached, with the final 15 cycles annealed at these final temperatures. Touch-down PCR has been shown to maximize the yield of product when using short primers (17 ). Amplifications with 16mer, 12mer and 10mer primers were analyzed by electrophoresis of 10 [mu]l of the reaction mixture on a 2% agarose gel and detection of the bands by ethidium bromide staining. Amplifications with 8mer and 6mer primers were analyzed by electrophoresis of 5 [mu]l of reaction mixture on an 8% polyacrylamide sequencing gel and detection of the bands by silver staining (Daiichi).
RESULTS AND DISCUSSION
Figure 1 shows the structure of the ODNs conjugated at their 5'-terminus to CDPI3. The preparation of these MGB-ODN conjugates has been previously reported by Lukhtanov et al. (15 ) and was accomplished by reaction of the 2,3,5,6-tetrafluorophenyl ester of CDPI3 with an oligonucleotide with a 5'-aminohexyl phosphate ester.
ACKNOWLEDGEMENTS
We wish to thank Dr Vladimir Gorn for oligonucleotide synthesis. A portion of this work was funded by grant GM 52774 from the National Institutes of Health, USPHS.
REFERENCES
1 Saiki,R.K. (1989) In Erlich,H.A. (ed.), PCR Technology. Principles and Applications for DNA Amplification. Stockton Press, pp. 7-16.
2 Tung,J.-S., Daugherty,B.L., O'Neill,L., Law,S.W., Han,J. and Mark,G.E. (1989) In Erlich,H.A. (ed.), PCR Technology. Principles and Applications for DNA Amplification. Stockton press, pp. 99-104.
