- Email: [email protected]
[2]Detection of point mutations by solid-phase minisequencing
[2]
Detection of Point Mutations by Solid-Phase Minisequencing Ann-Christine Syv~inen and Leena Peltonen
Introduction Many mutation types, including large alterations of chromosomal structures, rearrangements, extensions, deletions, or insertions of varying size, have been identified in the human genome. However, 95% of the identified disease-causing mutations are point mutations affecting one or a few nucleotides (1). Because genetic disorders rarely are caused by a single mutation per disease gene in a population, the ability to detect several mutations simultaneously per sample is of central importance. Consequently there is a need for reliable and technically simple methods for detecting point mutations both in clinical diagnostics and in research laboratories studying human genetic disorders. Another target for monitoring single-nucleotide changes is polymorphisms that have been estimated to occur on the average at 1 nucleotide out of 500 in the human genome (1). Analysis of this allelic variation can be utilized in tissue typing, in the identification of individuals, and in population genetic studies. In the diagnosis of infectious diseases, efficient methods for detecting sequence variants of genes are also required to identify virulent forms of microbes or resistance to drugs, as well as for typing bacteria and viruses in epidemiological studies. The methods currently used for detecting single-nucleotide variations rely in most cases on amplification of a DNA fragment spanning the variable nucleotide by the polymerase chain reaction (PCR) (2), which allows both specific and sensitive analysis of the target DNA sequence. Mutant and normal sequences can be distinguished using sequence-specific oligonucleotides either as hybridization probes or as primers in the PCR reaction, or by using nucleic acid-specific enzymes, such as restriction enzymes, DNA polymerases, or DNA ligases. Numerous modifications, combinations, and formats of the above-mentioned assay principles to distinguish between sequence variants have been developed (for reviews, see, e.g., Refs. 3 and 4). We have devised a convenient method, solid-phase minisequencing, for the detection of single-nucleotide variations, small deletions, or insertions in DNA fragments amplified by the PCR (5), and have applied it for detection of numerous diseasecausing mutations and single-nucleotide polymorphisms in human genes (for a summary see Ref. 6 and Table I). The major advantage of this method is that its high specificity allows unequivocal identification of any nucleotide variation under the same reaction conditions. Because the assay is carried out in a solid-phase format it comprises simple manipulations in a microtiter well or test tube format without gel-
14
Methods in Molecular Genetics, Volume 8 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
[2]
POINT MUTATION DETECTION BY MINISEQUENCING
TABLE I
15
Applications o f Solid-Phase Minisequencing Method .
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Application area Diagnosis of monogenic disorders Aspartylglucosaminuria Cystic fibrosis Familial amyloidosis of Finnish type
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Gene
Purpose
Ref. a
Aspartylglucosaminidase
Carrier screening Carrier screening Presymptomatic diagnosis Presymptomatic diagnosis Quantification of mutant transcripts Quantification of mutant transcripts
b c d
CFTR
Gelsolin
a~-Antitrypsin deficiency
a1-Antitrypsin
Marfan syndrome
Fibrillin
Congenital FXIII deficiency
Coagulation factor XIII
Diagnosis of mitochondrial diseases MERRF (myclonus with epilepsy and with ragged red fibers) MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) Leber's hereditary optic neuretinopathy NARP (neurogenic muscle weakness, ataxia, and retinis pigmeutosa)
f g
Diagnosis, quantification of heteroplasmy Diagnosis, quantification of heteroplasmy
h
Diagnosis, quantification of heteroplasmy Diagnosis, quantification of heteroplasmy
j
N-ras
Diagnosis, follow-up
1
Kras
Diagnosis
m
Apolipoprotein E Coagulation factor V Set of 12 biallelic markers Set of 12 biallelic markers
Allelic identification Allelic identification Forensic and paternity analyses Genetic mapping
n o p
tRNALy~ tRNALe,,
ND2, ND4
Subunit of ATP synthase
Identification of somatic mutations Acute myeloid leukemia, myelodysplastic syndromes Pancreatic carcinoma
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Identification of polymorphic nucleotides
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a The primer sequences and PCR conditions are given in these references. b A.-C. Syv~inen, E. Ikonen, T. Manninen, M. Bengstr6m, H. S6derlund, E Aula, and L. Peltonen, Genomics 12, 590(1992). c A. Jalanko, J. Kere, E. Savilahti, M. Schwartz, A.-C. Syv~inen,M. Ranki, and H. SOderlund, Clin. Chem. 38, 39 (1992). d T. Paunio, S. Kiuru, V. Hongell, E. Mustonen, A.-C. Syvfinen, M. Bengstr6m, J. Palo, and L. Peltonen, Genomics 13, 237 (1992). e L. Harju, T. Weber, L. Alexsandrova, M. Lukin, M. Ranki, and A. Jalanko, Clin. Chem. 2, 2282 (1993). f K. Kainulainen, L. Y. Sakai, A. Child, E M. Pope, L. Puhakka, L. Ryh~inen,A. Palotie, I. Kaitila, and L. Peltonen, Proc. Natl. Acad. Sci. U.S.A. 89, 5917 (1992). (continued)
16
I
MUTATION DETECTION IN H U M A N GENES
electrophoretic separation steps. Because the results of the assay are obtained as numeric values, their interpretation requires no special expertise and the numeric format facilitates computer-assisted handling of the data.
Principle of Method Figure 1 illustrates the principle of the solid-phase minisequencing method. A DNA fragment spanning the site of the mutation is first amplified using one biotinylated and one unbiotinylated PCR primer. The amplified DNA fragment carrying a biotin residue in the 5' end of one of its strands is captured on a solid support, taking advantage of the biotin-avidin interaction. The excess of unbiotinylated primer and nucleoside triphosphates (dNTPs) from PCR is removed by washing the solid support, and the unbiotinylated strand of the amplified fragment is removed by alkaline denaturation. The mutant and normal nucleotides are distinguished in the captured DNA strand by two separate "minisequencing" reactions. In a minisequencing reaction a DNA polymerase is used to specifically extend the 3' end of an oligonucleotide primer that anneals immediately upstream of a variable nucleotide position with a single labeled dNTP complementary to the nucleotide at the variable position. The incorporated labeled dNTP serves as a highly specific indicator of the nucleotide present at the variable site of the template. In samples from homozygous individuals a labeled dNTP will be incorporated in only one of the reactions and in samples from heterozygous individuals a dNTP will be incorporated in both reactions. After the minisequencing reaction the amount of incorporated label is measured and the ratio between the labels incorporated in the two reactions defines the genotype of the sample.
TABLE I
(continued)
g H. Mikkola, M. Syrj~il~i,V. Rasi, E. Vahtera, E. H~im~ilfiinen,L. Peltonen, and A. Palotie, Blood 84, 517 (1994). h A. Suomalainen, P. Kollmann, J.-N. Octave, H. S6derlund, and A.-C. Syv~inen, Eur. J. Hum. Genet. 1, 88 (1993). i A. Suomalainen, A. Majander, H. Pihko, L. Peltonen, and A.-C. Syv~inen, Hum. Mol. Genet. 2, 525 (1993). J g. Juvonen, K. Huoponen, A.-C. Syv~inen, P. Aula, E. Nikoskelainen, and M. Savontaus, Hum. Genet. 93, 16 (1994). k p. M~ikel~i-Bengs, A. Suomalainen, A. Majander, J. Rapola, H. Kalimo, A. Nuutila, and H. Pihko, Pediatr. Res. 37, 634 (1994). l A.-C. Syv~inen,H. S6derlund, E. Laaksonen, M. Bengtstr6m, M. Turunen, and A. Palotie, Int. J. Cancer 50, 713 (1992). m j. Ihalainen, M. Taavitsainen, T. Salmivaara, and A. Palotie, J. Clin. Pathol, 47, 1082 (1994). n A.-C. Syvfinen, K. Aalto-Set~ila, L. Harju, K. Kontula, and H. S6derlund, Genomics 8, 684 (1990). o A. Palotie, unpublished. P A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993). q J. Aaltonen, J. Komulainen, A. Wikman, A. Palotie, C. Wadelius, J. Peheentupa, and L. Peltonen, Eur. J. Hum. Genet. 1, 164 (1993).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING .
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FIG. 1 Steps of the solid-phase minisequencing method. (1) PCR with one biotinylated and one unbiotinylated primer. (2) Affinity-capture of the biotinylated PCR product in streptavidincoated microtiter wells. (3) Washing and denaturation. (4) The minisequencing primer extension reaction. (5) Measurement of the incorporated label. (6) Calculation of the result.
18
I
MUTATION DETECTION IN HUMAN GENES
P e r f o r m a n c e of Assay
Equipment and Reagents The following equipment is needed for carrying out the solid-phase minisequencing assay: access to oligonucleotide synthesis, programmable heat block for PCR, shaker at 37 ~C, water bath for incubation at 50~ liquid scintillation counter, and optionally, multichannel pipette or microtiter plate washer. Required materials and reagents include the following: thermostable DNA polymerase for PCR and for the minisequencing reaction (Taq DNA polymerase; Promega Biotech, Madison, WI), 3H-labeled deoxynucleoside triphosphates (dATE TRK 625; dCTP, TRK 576; dGTE TRK 627; dTTP, TRK 633; Amersham, Arlington Heights, IL), streptavidin-coated microtiter plates (Combiplate 8; Labsystems, Helsinki, Finland), and biotinylphosphoramidite reagent for biotinylation of one of the PCR primers (RPN 2012; Amersham). Reagents and materials from sources other than those indicated here can also be used. Reagents of the highest purity grade are used for preparation of buffers and other solutions.
Design of Primers A 5' and a 3' PCR primer and a detection step primer for the minisequencing reaction are required. The PCR primers should yield an amplification product spanning the variable nucleotide position preferably between 80 and 200 base pairs (bp) in size. The PCR primers should be 20-23 nucleotides long and have similar melting temperatures and noncomplementary 3' ends (7). The 5' end of one of the PCR primers is biotinylated in the last step of the synthesis. The minisequencing detection step primer should be 20 nucleotides long and complementary to the biotinylated strand of the PCR product immediately 3' of the variable nucleotide position (Fig. 2). To detect a deletion or an insertion, the first nucleotide of the deletion/insertion should differ from the first nucleotide following the deletion/insertion.
Procedure The steps are numbered as in Fig. 1.
Step 1: Polymerase Chain Reaction Any type of DNA sample, treated as is suitable for PCR amplification (8), can be analyzed. RNA is amplified after synthesis of a first-strand cDNA by reverse transcriptase (9).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING
A
19
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normal ATCATC"* TAGTAGAAACCAC
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FIG. 2 Example of detection of (A) a G-to-C transversion and (B) a 3-bp (CTT) deletion by solid-phase minisequencing. Bio, Biotinylated primer; the variable nucleotide and the incorporated labeled nucleotide are indicated in boldface type; asterisk (*) symbolizes the label on the incorporated nucleotide; and the five 3' nucleotides of the minisequencing detection step primer annealing immediately next to the variable nucleotide position are shown.
The PCR is most conveniently carried out in a 50-#1 volume. Prepare a master mix containing 5 #1 of 10 X concentrated Taq DNA polymerase buffer [500 mM TrisHC1 (pH 8.8), 150 mM (NH4)2SO4, 15 mM MgCI2, 7% (v/v) Triton X-100, 0.7% (w/v) gelatin], 5 #1 of a mixture of all four dNTS at 2 mM concentration, and dTTP, 2.5 #1 of 20 # M unbiotinylated PCR primer, 2.5/~1 of 4 # M biotinylated primer, and distilled water to 35 #l/reaction. Add 35/~1 of the PCR reagent mixture to 10 pJ of a solution containing 30 to 3 X 104 molecules of the DNA template. Initiate the PCR by a "hot start" by first incubating the samples in a programmable heat block for 3 min at 95 ~C, followed by addition of 1.25 units of Taq DNA polymerase in 5/1,1 at 80 ~C. Carry out 30 PCR cycles of 1 min at 95 ~C, 1 min at an annealing temperature (which is determined by the sequence of the primers, usually between 50 and 60 ~C), and 1 min at 72 ~C. Comment Precautions for avoiding DNA contamination in the PCR should be taken (10). Obviously other PCR conditions than those given above can be applied. However, two points of importance for the so]id-phase minisequencing method should be considered.
20
I
MUTATION DETECTION IN HUMAN GENES
1. The biotin-binding capacity of the streptavidin-coated microtiter wells used to capture the biotinylated PCR products (and excess of biotinylated PCR primer) sets an upper limit to the amount of biotinylated primer that can be used in the PCR. The biotin-binding capacity of the streptavidin-coated wells we use is about 2 pmol. Therefore, in the standard protocol we use 10 pmol of the biotinylated primer per PCR reaction and analyze one-fifth of the PCR product per minisequencing reaction. If "multiplex" PCR is used for amplification of more than one locus, a smaller aliquot of the PCR product should be analyzed. For multiplex PCRs of four loci we found it optimal to detect the polymorphic nucleotides in ~o of the PCR product per well (11). Avidin- or streptavidin-coated microparticles with significantly higher biotin-binding capacity than microtiter plates can also be used as solid support (12). 2. A prerequisite for successful use of the solid-phase minisequencing method with 3H (which has a low specific activity) as label is that the PCR amplification is efficient. Ten microliters of the PCR product should be clearly visible in an agarose gel by staining with ethidium bromide.
Step 2: Capturing Reaction Transfer two 10-/xl aliquots of the amplified sample to streptavidin-coated microtiter wells. Include negative controls containing 10/zl of Taq DNA polymerase buffer for both minisequencing reactions. Add 40/xl of 20 mM sodium phosphate buffer (pH 7.5), 100 mM NaC1, and 0.1% (v/v) Tween 20 to each well. Seal the wells with a sticker and incubate the microtiter plate for 1.5 hr at 37~ with gentle shaking. Discard the contents of the wells. Wash the wells at room temperature three times by adding 200/zl of washing solution [40 mM Tris-HC1 (pH 8.8), 1 mM EDTA, 50 mM NaC1, 0.1% (v/v) Tween 20]. Empty the wells thoroughly between the washes. Comment It is important for the specificity of the minisequencing reaction that the excess of dNTPs present during the PCR are completely removed by the washing steps. The use of an automatic microtiter plate washer saves time and improves the washing efficiency.
Step 3: Denaturation Add 100/zl of 50 mM NaOH to each well, and incubate at room temperature for 2 - 5 min. Discard the contents of the wells and wash as described in step 2.
Step 4: Minisequencing Reaction Prepare two master mixes, one for detection of the mutant nucleotide and the other for detection of the normal nucleotide, by combining 5/xl of 10 x Taq DNA polymerase buffer (see step 1 above), 2/zl of 5/zM detection step primer, 0.1/zCi (usually 0.1/zl) of 3H-labeled dNTP complementary to the nucleotide to be detected, 0.1 unit of Taq DNA polymerase, and distilled water to 50/xl/reaction. Prepare the master
[2] POINT MUTATION DETECTION BY MINISEQUENCING
21
mix during the capturing reaction and store at room temperature until use. For each sample, add 50 #1 of minisequencing reaction mix for detection of the mutant nucleotide to one well and 50 #1 of reaction mix for detection of the normal nucleotide to another well. Incubate at 50~ for 10 min. Discard the contents of the wells and wash as described in step 2. Comment 1. The reaction conditions for annealing the primer to the immobilized DNA strand are nonstringent. Therefore, the same reaction conditions can be used for analysis of any DNA fragment, irrespectively of the nucleotide sequence of the detection step primer. 2. An advantage of using a thermostable polymerase for the minisequencing primer extension reaction is that annealing of the primer to the template and extension of the primer with the labeled dNTP can be carried out simultaneously at a fairly high temperature (50 ~C) that is favorable for both reactions. 3. Deoxynucleoside triphosphates labeled with other radioisotopes (32p or 355) (5) or with haptens (13) can also be used.
Step 5: Measurement of lncorporated Label Release the primer after the minisequencing reaction by incubating the microtiter well with 60 #1 of 50 mM NaOH for 2 - 5 min at room temperature. Transfer the eluted primer to scintillation vials, add scintillation fluid, and measure the eluted 3H in a liquid scintillation counter. Comment By using streptavidin-coated microtiter plates manufactured from scintillating polystyrene (ScintiStrips; Wallac OY, Turku, Finland) as solid support, the final washing and denaturation steps and the transfer of the eluted primer to scintillation vials can be omitted (14). This requires a scintillation counter for microtiter plates.
Step 6: Interpretation of Result Calculate the ratio (R value) between the 3H-labeled dNTP incorporated in the reaction for detecting the mutant nucleotide and the 3H-labeled dNTP incorporated in the reaction for detecting the normal nucleotide. The R value will be > 10 in samples from individuals homozygous for the mutant nucleotide, <0.1 in samples from individuals homozygous for the normal nucleotide, and in samples from heterozygous individuals it will usually be between 0.5 and 2.0, depending on the specific activities of the 3H-labeled dNTPs used (Table II). Comment Calculation of the R value eliminates variations in the amount of incorporated 3Hlabeled dNTPs due to sample-to-sample variations in the efficiency of the PCR am-
22 TABLE II
I
MUTATION DETECTION IN H U M A N GENES
D e t e c t i o n o f P o l y m o r p h i c N u c l e o t i d e s (A > G) in the A D H 3 and M E T H G e n e s by Solid-Phase Minisequencing ADH3
METH
[3H]dNTP incorporated (cpm) a
[3H]dNTP incorporated (cpm) a R value
Sample 1 2 3
A allele
G allele
(Acpm/Gcpm)
Genotype
A allele
G allele
R value (Acpm/Gcpm)
Genotype
6550 160 2610
61 2800 1470
107 0.057 1.77
AA GG AG
53 970 1900
4570 1530 105
0.011 0.63 18.0
GG AG AA
30
39
27
38
H20
m
a At both sites, one [3H]dATP will be incorporated in the A allele. At the ADH3 site, one [3H]dGTP will be incorporated, and at the METH site three [3H]dGTPs will be incorporated in the G allele. In the analysis of the ADH3 locus the specific activities of [3H]dATP and [3H]dGTP were 62 and 31 Ci/mmol, respectively. In the analysis of the METH locus the corresponding specific activities were 61 and 40 Ci/mmol. [Data from A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993).]
plification. If the sequence contains one (or more) identical nucleotides immediately next to the nucleotide at the variable site, one (or more) additional [3H]dNTPs will be incorporated in the minisequencing reaction, which obviously affects the R value. Table II shows as an example the results from analyzing polymorphic nucleotides in the alcohol dehydrogenase gene and the METH protooncogene, in which one or three 3H-labeled dGTPs are incorporated, respectively. The small sequence-specific background misincorporation by the Taq DNA polymerase, which most probably is due to other dNTPs present as impurities in the 3H-labeled dNTPs, has only a minor effect on the R value.
Discussion In addition to unequivocally distinguishing between two sequences present either in homozygous (allele ratio 2:0) or heterozygous (allele ratio 1 : 1) samples, the R value obtained in the solid-phase minisequencing method directly reflects the ratio between two sequences when they are present in a sample as a mixture in any other ratio. Because the two sequences are essentially identical, they are amplified with equal efficiency during the PCR, which results in a linear relationship between the R value obtained in the minisequencing assay and the initial ratio between the two sequences (Fig. 3). Consequently, the method is a useful tool for quantitative PCR analysis. It is highly sensitive, allowing the detection of one sequence present as a small minority (< 1%) in a sample ( 15, 16).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING
23
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FIG. 3 Solid-phase minisequencing standard curve. Mixtures of known amounts of two 63mer oligonucleotides differing from each other at a single nucleotide (nucleotide 3243 of the mitochondrial tRNALeugene; Ref. 16) were analyzed and the R values obtained in the minisequencing assay were plotted as a function of the initial ratio between the two sequences. The R values obtained by analyzing each oligonucleotide separately are indicated by the horizontal bars. The R values are the means of three parallel assays.
We have utilized the quantitative character of the solid-phase minisequencing method to analyze from pooled DNA samples two sequences present as a mixture in this DNA sample to determine population frequencies of disease-causing mutant alleles and polymorphisms (11, 16) as well as to determine the proportion of heteroplasmic mutations of the mitochondrial DNA (17-19). To determine the absolute amount of a nucleic acid sequence present in a sample, a known amount of internal standard differing from the sequence to be quantified by a single nucleotide is added to the sample before the PCR amplification (18, 20). Genomic DNA of known genotype (16), synthetic oligonucleotides (18), and RNA prepared by in vitro transcription (20) have been shown to be suitable for use as internal standards. The initial ratio between the two amplified sequences can be calculated directly from the obtained R value, taking into account the specific activities and number of 3H-labeled dNTPs incorporated in the minisequencing reactions. Alternatively, the initial ratio between the two sequences can be determined by comparing the obtained R value with a standard curve prepared in parallel with mixtures of known amounts of the corresponding sequences. The use of a standard curve will correct for possible misincorporation of an [3H]dNTP by the DNA polymerase, which may affect the result when a sequence present as a small minority of the other sequence is to be quantified.
24
I MUTATIONDETECTION IN HUMAN GENES In conclusion, the high specificity of the single-nucleotide primer extension reaction catalyzed by a DNA polymerase is utilized in the minisequencing method, and this allows both unequivocal discrimination between sequence variants and sensitive quantitative PCR analysis. The combination of this assay principle with a solid-phase format yields a robust assay that is both suitable for large-scale use (21) and easy to set up for detecting single-nucleotide variations for different purposes both in the research laboratory and in the routine clinical laboratory.
References 1. V. A. McKusick, in "Mendelian Inheritance of Man," Vol. 1, 10th ed., p. xxxi. The Johns Hopkins University Press, Baltimore, Maryland, 1992. 2. K. B. Mullis and E A. Faloona, Methods Enzymol. 155, 335 (1987). 3. R. G. H. Cotton, Mutat. Res. 285, 125 (1993). 4. A.-C. Syv~inen and U. Landegren, Hum. Mutat. 3, 172 (1994). 5. A.-C. Syv~inen, K. Aalto-Set~il~i, L. Harju, K. Kontula, and H. S6derlund, Genomics 8, 684 (1990). 6. A.-C. Syv~inen, Clin. Chim. Acta 226, 225, (1994). 7. M. A. Innis and D. H. Gelfand, in "PCR Protocols. A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 3. Academic Press, San Diego, California, 1990. 8. R. Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification" (H. A. Erlich, ed.), p. 35. Stockton Press, New York, 1989. 9. E. S. Kawasaki, in "PCR Protocols. A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 21. Academic Press, San Diego, California, 1990. 10. C. Orrego, in "PCR Protocols: A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 451. Academic Press, San Diego, California, 1990. 11. A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993). 12. A.-C. Syv~inen and H. S6dedund, Methods Enzymol. 218, 474 (1993). 13. L. Harju, T. Weber, L. Alexsandrova, M. Lukin, M. Ranki, and A. Jalanko, Clin. Chem. 2, 2282 (1993). 14. J. Ihalainen, H. Siitari, S. Laine, A.-C. Syv~inen, and A. Palotie, BioTechniques 16, 938 (1994). 15. A.-C. Syv~en, H. S6dedund, E. Laaksonen, M. Bengtstr6m, M. Turunen, and A. Palotie, Int. J. Cancer50, 713 (1992). 16. A.-C. Syv~en, E. Ikonen, T. Manninen, M. Bengstr6m, H. S6derlund, E Aula, and L. Peltonen, Genomics 12, 590 (1992). 17. A. Suomalainen, E Kollmann, J.-N. Octave, H. S6dedund, and A.-C. Syv~inen, Eur. J. Hum. Genet. 1, 88 (1993). 18. A. Suomalainen, A. Majander, H. Pihko, L. Peltonen, and A.-C. Syv~inen, Hum. Mol. Genet. 2, 525 (1993).
[2] POINTMUTATION DETECTION BY MINISEQUENCING
25
19. V. Juvonen, K. Huoponen, A.-C. Syv~inen, E Aula, E. Nikoskelainen, and M. Savontaus, Hum. Genet. 93, 16 (1994). 20. E. Ikonen, T. Manninen, L. Peltonen, and A.-C. Syv~inen, PCR Methods Appl. 1, 234 (1992). 21. M. Hietala, H. Gr6n, A.-C. Syv~inen, L. Peltonen, and E Aula, Eur. J. Hum. Genet. 1, 296 (1993).
Detection of Point Mutations by Solid-Phase Minisequencing Ann-Christine Syv~inen and Leena Peltonen
Introduction Many mutation types, including large alterations of chromosomal structures, rearrangements, extensions, deletions, or insertions of varying size, have been identified in the human genome. However, 95% of the identified disease-causing mutations are point mutations affecting one or a few nucleotides (1). Because genetic disorders rarely are caused by a single mutation per disease gene in a population, the ability to detect several mutations simultaneously per sample is of central importance. Consequently there is a need for reliable and technically simple methods for detecting point mutations both in clinical diagnostics and in research laboratories studying human genetic disorders. Another target for monitoring single-nucleotide changes is polymorphisms that have been estimated to occur on the average at 1 nucleotide out of 500 in the human genome (1). Analysis of this allelic variation can be utilized in tissue typing, in the identification of individuals, and in population genetic studies. In the diagnosis of infectious diseases, efficient methods for detecting sequence variants of genes are also required to identify virulent forms of microbes or resistance to drugs, as well as for typing bacteria and viruses in epidemiological studies. The methods currently used for detecting single-nucleotide variations rely in most cases on amplification of a DNA fragment spanning the variable nucleotide by the polymerase chain reaction (PCR) (2), which allows both specific and sensitive analysis of the target DNA sequence. Mutant and normal sequences can be distinguished using sequence-specific oligonucleotides either as hybridization probes or as primers in the PCR reaction, or by using nucleic acid-specific enzymes, such as restriction enzymes, DNA polymerases, or DNA ligases. Numerous modifications, combinations, and formats of the above-mentioned assay principles to distinguish between sequence variants have been developed (for reviews, see, e.g., Refs. 3 and 4). We have devised a convenient method, solid-phase minisequencing, for the detection of single-nucleotide variations, small deletions, or insertions in DNA fragments amplified by the PCR (5), and have applied it for detection of numerous diseasecausing mutations and single-nucleotide polymorphisms in human genes (for a summary see Ref. 6 and Table I). The major advantage of this method is that its high specificity allows unequivocal identification of any nucleotide variation under the same reaction conditions. Because the assay is carried out in a solid-phase format it comprises simple manipulations in a microtiter well or test tube format without gel-
14
Methods in Molecular Genetics, Volume 8 Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
[2]
POINT MUTATION DETECTION BY MINISEQUENCING
TABLE I
15
Applications o f Solid-Phase Minisequencing Method .
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Application area Diagnosis of monogenic disorders Aspartylglucosaminuria Cystic fibrosis Familial amyloidosis of Finnish type
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Purpose
Ref. a
Aspartylglucosaminidase
Carrier screening Carrier screening Presymptomatic diagnosis Presymptomatic diagnosis Quantification of mutant transcripts Quantification of mutant transcripts
b c d
CFTR
Gelsolin
a~-Antitrypsin deficiency
a1-Antitrypsin
Marfan syndrome
Fibrillin
Congenital FXIII deficiency
Coagulation factor XIII
Diagnosis of mitochondrial diseases MERRF (myclonus with epilepsy and with ragged red fibers) MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) Leber's hereditary optic neuretinopathy NARP (neurogenic muscle weakness, ataxia, and retinis pigmeutosa)
f g
Diagnosis, quantification of heteroplasmy Diagnosis, quantification of heteroplasmy
h
Diagnosis, quantification of heteroplasmy Diagnosis, quantification of heteroplasmy
j
N-ras
Diagnosis, follow-up
1
Kras
Diagnosis
m
Apolipoprotein E Coagulation factor V Set of 12 biallelic markers Set of 12 biallelic markers
Allelic identification Allelic identification Forensic and paternity analyses Genetic mapping
n o p
tRNALy~ tRNALe,,
ND2, ND4
Subunit of ATP synthase
Identification of somatic mutations Acute myeloid leukemia, myelodysplastic syndromes Pancreatic carcinoma
e
i
k
Identification of polymorphic nucleotides
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a The primer sequences and PCR conditions are given in these references. b A.-C. Syv~inen, E. Ikonen, T. Manninen, M. Bengstr6m, H. S6derlund, E Aula, and L. Peltonen, Genomics 12, 590(1992). c A. Jalanko, J. Kere, E. Savilahti, M. Schwartz, A.-C. Syv~inen,M. Ranki, and H. SOderlund, Clin. Chem. 38, 39 (1992). d T. Paunio, S. Kiuru, V. Hongell, E. Mustonen, A.-C. Syvfinen, M. Bengstr6m, J. Palo, and L. Peltonen, Genomics 13, 237 (1992). e L. Harju, T. Weber, L. Alexsandrova, M. Lukin, M. Ranki, and A. Jalanko, Clin. Chem. 2, 2282 (1993). f K. Kainulainen, L. Y. Sakai, A. Child, E M. Pope, L. Puhakka, L. Ryh~inen,A. Palotie, I. Kaitila, and L. Peltonen, Proc. Natl. Acad. Sci. U.S.A. 89, 5917 (1992). (continued)
16
I
MUTATION DETECTION IN H U M A N GENES
electrophoretic separation steps. Because the results of the assay are obtained as numeric values, their interpretation requires no special expertise and the numeric format facilitates computer-assisted handling of the data.
Principle of Method Figure 1 illustrates the principle of the solid-phase minisequencing method. A DNA fragment spanning the site of the mutation is first amplified using one biotinylated and one unbiotinylated PCR primer. The amplified DNA fragment carrying a biotin residue in the 5' end of one of its strands is captured on a solid support, taking advantage of the biotin-avidin interaction. The excess of unbiotinylated primer and nucleoside triphosphates (dNTPs) from PCR is removed by washing the solid support, and the unbiotinylated strand of the amplified fragment is removed by alkaline denaturation. The mutant and normal nucleotides are distinguished in the captured DNA strand by two separate "minisequencing" reactions. In a minisequencing reaction a DNA polymerase is used to specifically extend the 3' end of an oligonucleotide primer that anneals immediately upstream of a variable nucleotide position with a single labeled dNTP complementary to the nucleotide at the variable position. The incorporated labeled dNTP serves as a highly specific indicator of the nucleotide present at the variable site of the template. In samples from homozygous individuals a labeled dNTP will be incorporated in only one of the reactions and in samples from heterozygous individuals a dNTP will be incorporated in both reactions. After the minisequencing reaction the amount of incorporated label is measured and the ratio between the labels incorporated in the two reactions defines the genotype of the sample.
TABLE I
(continued)
g H. Mikkola, M. Syrj~il~i,V. Rasi, E. Vahtera, E. H~im~ilfiinen,L. Peltonen, and A. Palotie, Blood 84, 517 (1994). h A. Suomalainen, P. Kollmann, J.-N. Octave, H. S6derlund, and A.-C. Syv~inen, Eur. J. Hum. Genet. 1, 88 (1993). i A. Suomalainen, A. Majander, H. Pihko, L. Peltonen, and A.-C. Syv~inen, Hum. Mol. Genet. 2, 525 (1993). J g. Juvonen, K. Huoponen, A.-C. Syv~inen, P. Aula, E. Nikoskelainen, and M. Savontaus, Hum. Genet. 93, 16 (1994). k p. M~ikel~i-Bengs, A. Suomalainen, A. Majander, J. Rapola, H. Kalimo, A. Nuutila, and H. Pihko, Pediatr. Res. 37, 634 (1994). l A.-C. Syv~inen,H. S6derlund, E. Laaksonen, M. Bengtstr6m, M. Turunen, and A. Palotie, Int. J. Cancer 50, 713 (1992). m j. Ihalainen, M. Taavitsainen, T. Salmivaara, and A. Palotie, J. Clin. Pathol, 47, 1082 (1994). n A.-C. Syvfinen, K. Aalto-Set~ila, L. Harju, K. Kontula, and H. S6derlund, Genomics 8, 684 (1990). o A. Palotie, unpublished. P A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993). q J. Aaltonen, J. Komulainen, A. Wikman, A. Palotie, C. Wadelius, J. Peheentupa, and L. Peltonen, Eur. J. Hum. Genet. 1, 164 (1993).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING .
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FIG. 1 Steps of the solid-phase minisequencing method. (1) PCR with one biotinylated and one unbiotinylated primer. (2) Affinity-capture of the biotinylated PCR product in streptavidincoated microtiter wells. (3) Washing and denaturation. (4) The minisequencing primer extension reaction. (5) Measurement of the incorporated label. (6) Calculation of the result.
18
I
MUTATION DETECTION IN HUMAN GENES
P e r f o r m a n c e of Assay
Equipment and Reagents The following equipment is needed for carrying out the solid-phase minisequencing assay: access to oligonucleotide synthesis, programmable heat block for PCR, shaker at 37 ~C, water bath for incubation at 50~ liquid scintillation counter, and optionally, multichannel pipette or microtiter plate washer. Required materials and reagents include the following: thermostable DNA polymerase for PCR and for the minisequencing reaction (Taq DNA polymerase; Promega Biotech, Madison, WI), 3H-labeled deoxynucleoside triphosphates (dATE TRK 625; dCTP, TRK 576; dGTE TRK 627; dTTP, TRK 633; Amersham, Arlington Heights, IL), streptavidin-coated microtiter plates (Combiplate 8; Labsystems, Helsinki, Finland), and biotinylphosphoramidite reagent for biotinylation of one of the PCR primers (RPN 2012; Amersham). Reagents and materials from sources other than those indicated here can also be used. Reagents of the highest purity grade are used for preparation of buffers and other solutions.
Design of Primers A 5' and a 3' PCR primer and a detection step primer for the minisequencing reaction are required. The PCR primers should yield an amplification product spanning the variable nucleotide position preferably between 80 and 200 base pairs (bp) in size. The PCR primers should be 20-23 nucleotides long and have similar melting temperatures and noncomplementary 3' ends (7). The 5' end of one of the PCR primers is biotinylated in the last step of the synthesis. The minisequencing detection step primer should be 20 nucleotides long and complementary to the biotinylated strand of the PCR product immediately 3' of the variable nucleotide position (Fig. 2). To detect a deletion or an insertion, the first nucleotide of the deletion/insertion should differ from the first nucleotide following the deletion/insertion.
Procedure The steps are numbered as in Fig. 1.
Step 1: Polymerase Chain Reaction Any type of DNA sample, treated as is suitable for PCR amplification (8), can be analyzed. RNA is amplified after synthesis of a first-strand cDNA by reverse transcriptase (9).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING
A
19
normal 5'- Bio
GAATTGCCAGC ,....CGGTCG
mutation G---* C 5'- Bio
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GAATTCCCAGC #=-~GGTCG
normal ATCATC"* TAGTAGAAACCAC
Bio- 5'
ATCATT" * TAGTAACCAC
Bio- 5'
_
_
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mutation del CTT
FIG. 2 Example of detection of (A) a G-to-C transversion and (B) a 3-bp (CTT) deletion by solid-phase minisequencing. Bio, Biotinylated primer; the variable nucleotide and the incorporated labeled nucleotide are indicated in boldface type; asterisk (*) symbolizes the label on the incorporated nucleotide; and the five 3' nucleotides of the minisequencing detection step primer annealing immediately next to the variable nucleotide position are shown.
The PCR is most conveniently carried out in a 50-#1 volume. Prepare a master mix containing 5 #1 of 10 X concentrated Taq DNA polymerase buffer [500 mM TrisHC1 (pH 8.8), 150 mM (NH4)2SO4, 15 mM MgCI2, 7% (v/v) Triton X-100, 0.7% (w/v) gelatin], 5 #1 of a mixture of all four dNTS at 2 mM concentration, and dTTP, 2.5 #1 of 20 # M unbiotinylated PCR primer, 2.5/~1 of 4 # M biotinylated primer, and distilled water to 35 #l/reaction. Add 35/~1 of the PCR reagent mixture to 10 pJ of a solution containing 30 to 3 X 104 molecules of the DNA template. Initiate the PCR by a "hot start" by first incubating the samples in a programmable heat block for 3 min at 95 ~C, followed by addition of 1.25 units of Taq DNA polymerase in 5/1,1 at 80 ~C. Carry out 30 PCR cycles of 1 min at 95 ~C, 1 min at an annealing temperature (which is determined by the sequence of the primers, usually between 50 and 60 ~C), and 1 min at 72 ~C. Comment Precautions for avoiding DNA contamination in the PCR should be taken (10). Obviously other PCR conditions than those given above can be applied. However, two points of importance for the so]id-phase minisequencing method should be considered.
20
I
MUTATION DETECTION IN HUMAN GENES
1. The biotin-binding capacity of the streptavidin-coated microtiter wells used to capture the biotinylated PCR products (and excess of biotinylated PCR primer) sets an upper limit to the amount of biotinylated primer that can be used in the PCR. The biotin-binding capacity of the streptavidin-coated wells we use is about 2 pmol. Therefore, in the standard protocol we use 10 pmol of the biotinylated primer per PCR reaction and analyze one-fifth of the PCR product per minisequencing reaction. If "multiplex" PCR is used for amplification of more than one locus, a smaller aliquot of the PCR product should be analyzed. For multiplex PCRs of four loci we found it optimal to detect the polymorphic nucleotides in ~o of the PCR product per well (11). Avidin- or streptavidin-coated microparticles with significantly higher biotin-binding capacity than microtiter plates can also be used as solid support (12). 2. A prerequisite for successful use of the solid-phase minisequencing method with 3H (which has a low specific activity) as label is that the PCR amplification is efficient. Ten microliters of the PCR product should be clearly visible in an agarose gel by staining with ethidium bromide.
Step 2: Capturing Reaction Transfer two 10-/xl aliquots of the amplified sample to streptavidin-coated microtiter wells. Include negative controls containing 10/zl of Taq DNA polymerase buffer for both minisequencing reactions. Add 40/xl of 20 mM sodium phosphate buffer (pH 7.5), 100 mM NaC1, and 0.1% (v/v) Tween 20 to each well. Seal the wells with a sticker and incubate the microtiter plate for 1.5 hr at 37~ with gentle shaking. Discard the contents of the wells. Wash the wells at room temperature three times by adding 200/zl of washing solution [40 mM Tris-HC1 (pH 8.8), 1 mM EDTA, 50 mM NaC1, 0.1% (v/v) Tween 20]. Empty the wells thoroughly between the washes. Comment It is important for the specificity of the minisequencing reaction that the excess of dNTPs present during the PCR are completely removed by the washing steps. The use of an automatic microtiter plate washer saves time and improves the washing efficiency.
Step 3: Denaturation Add 100/zl of 50 mM NaOH to each well, and incubate at room temperature for 2 - 5 min. Discard the contents of the wells and wash as described in step 2.
Step 4: Minisequencing Reaction Prepare two master mixes, one for detection of the mutant nucleotide and the other for detection of the normal nucleotide, by combining 5/xl of 10 x Taq DNA polymerase buffer (see step 1 above), 2/zl of 5/zM detection step primer, 0.1/zCi (usually 0.1/zl) of 3H-labeled dNTP complementary to the nucleotide to be detected, 0.1 unit of Taq DNA polymerase, and distilled water to 50/xl/reaction. Prepare the master
[2] POINT MUTATION DETECTION BY MINISEQUENCING
21
mix during the capturing reaction and store at room temperature until use. For each sample, add 50 #1 of minisequencing reaction mix for detection of the mutant nucleotide to one well and 50 #1 of reaction mix for detection of the normal nucleotide to another well. Incubate at 50~ for 10 min. Discard the contents of the wells and wash as described in step 2. Comment 1. The reaction conditions for annealing the primer to the immobilized DNA strand are nonstringent. Therefore, the same reaction conditions can be used for analysis of any DNA fragment, irrespectively of the nucleotide sequence of the detection step primer. 2. An advantage of using a thermostable polymerase for the minisequencing primer extension reaction is that annealing of the primer to the template and extension of the primer with the labeled dNTP can be carried out simultaneously at a fairly high temperature (50 ~C) that is favorable for both reactions. 3. Deoxynucleoside triphosphates labeled with other radioisotopes (32p or 355) (5) or with haptens (13) can also be used.
Step 5: Measurement of lncorporated Label Release the primer after the minisequencing reaction by incubating the microtiter well with 60 #1 of 50 mM NaOH for 2 - 5 min at room temperature. Transfer the eluted primer to scintillation vials, add scintillation fluid, and measure the eluted 3H in a liquid scintillation counter. Comment By using streptavidin-coated microtiter plates manufactured from scintillating polystyrene (ScintiStrips; Wallac OY, Turku, Finland) as solid support, the final washing and denaturation steps and the transfer of the eluted primer to scintillation vials can be omitted (14). This requires a scintillation counter for microtiter plates.
Step 6: Interpretation of Result Calculate the ratio (R value) between the 3H-labeled dNTP incorporated in the reaction for detecting the mutant nucleotide and the 3H-labeled dNTP incorporated in the reaction for detecting the normal nucleotide. The R value will be > 10 in samples from individuals homozygous for the mutant nucleotide, <0.1 in samples from individuals homozygous for the normal nucleotide, and in samples from heterozygous individuals it will usually be between 0.5 and 2.0, depending on the specific activities of the 3H-labeled dNTPs used (Table II). Comment Calculation of the R value eliminates variations in the amount of incorporated 3Hlabeled dNTPs due to sample-to-sample variations in the efficiency of the PCR am-
22 TABLE II
I
MUTATION DETECTION IN H U M A N GENES
D e t e c t i o n o f P o l y m o r p h i c N u c l e o t i d e s (A > G) in the A D H 3 and M E T H G e n e s by Solid-Phase Minisequencing ADH3
METH
[3H]dNTP incorporated (cpm) a
[3H]dNTP incorporated (cpm) a R value
Sample 1 2 3
A allele
G allele
(Acpm/Gcpm)
Genotype
A allele
G allele
R value (Acpm/Gcpm)
Genotype
6550 160 2610
61 2800 1470
107 0.057 1.77
AA GG AG
53 970 1900
4570 1530 105
0.011 0.63 18.0
GG AG AA
30
39
27
38
H20
m
a At both sites, one [3H]dATP will be incorporated in the A allele. At the ADH3 site, one [3H]dGTP will be incorporated, and at the METH site three [3H]dGTPs will be incorporated in the G allele. In the analysis of the ADH3 locus the specific activities of [3H]dATP and [3H]dGTP were 62 and 31 Ci/mmol, respectively. In the analysis of the METH locus the corresponding specific activities were 61 and 40 Ci/mmol. [Data from A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993).]
plification. If the sequence contains one (or more) identical nucleotides immediately next to the nucleotide at the variable site, one (or more) additional [3H]dNTPs will be incorporated in the minisequencing reaction, which obviously affects the R value. Table II shows as an example the results from analyzing polymorphic nucleotides in the alcohol dehydrogenase gene and the METH protooncogene, in which one or three 3H-labeled dGTPs are incorporated, respectively. The small sequence-specific background misincorporation by the Taq DNA polymerase, which most probably is due to other dNTPs present as impurities in the 3H-labeled dNTPs, has only a minor effect on the R value.
Discussion In addition to unequivocally distinguishing between two sequences present either in homozygous (allele ratio 2:0) or heterozygous (allele ratio 1 : 1) samples, the R value obtained in the solid-phase minisequencing method directly reflects the ratio between two sequences when they are present in a sample as a mixture in any other ratio. Because the two sequences are essentially identical, they are amplified with equal efficiency during the PCR, which results in a linear relationship between the R value obtained in the minisequencing assay and the initial ratio between the two sequences (Fig. 3). Consequently, the method is a useful tool for quantitative PCR analysis. It is highly sensitive, allowing the detection of one sequence present as a small minority (< 1%) in a sample ( 15, 16).
[2] POINTMUTATIONDETECTIONBY MINISEQUENCING
23
R-value
100 -
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10-
1.0
-
0,1
-
0.01
-
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'
0.01
,
0.1
.....i"
1.0
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..............
10
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100
mutant sequence / normal sequence
FIG. 3 Solid-phase minisequencing standard curve. Mixtures of known amounts of two 63mer oligonucleotides differing from each other at a single nucleotide (nucleotide 3243 of the mitochondrial tRNALeugene; Ref. 16) were analyzed and the R values obtained in the minisequencing assay were plotted as a function of the initial ratio between the two sequences. The R values obtained by analyzing each oligonucleotide separately are indicated by the horizontal bars. The R values are the means of three parallel assays.
We have utilized the quantitative character of the solid-phase minisequencing method to analyze from pooled DNA samples two sequences present as a mixture in this DNA sample to determine population frequencies of disease-causing mutant alleles and polymorphisms (11, 16) as well as to determine the proportion of heteroplasmic mutations of the mitochondrial DNA (17-19). To determine the absolute amount of a nucleic acid sequence present in a sample, a known amount of internal standard differing from the sequence to be quantified by a single nucleotide is added to the sample before the PCR amplification (18, 20). Genomic DNA of known genotype (16), synthetic oligonucleotides (18), and RNA prepared by in vitro transcription (20) have been shown to be suitable for use as internal standards. The initial ratio between the two amplified sequences can be calculated directly from the obtained R value, taking into account the specific activities and number of 3H-labeled dNTPs incorporated in the minisequencing reactions. Alternatively, the initial ratio between the two sequences can be determined by comparing the obtained R value with a standard curve prepared in parallel with mixtures of known amounts of the corresponding sequences. The use of a standard curve will correct for possible misincorporation of an [3H]dNTP by the DNA polymerase, which may affect the result when a sequence present as a small minority of the other sequence is to be quantified.
24
I MUTATIONDETECTION IN HUMAN GENES In conclusion, the high specificity of the single-nucleotide primer extension reaction catalyzed by a DNA polymerase is utilized in the minisequencing method, and this allows both unequivocal discrimination between sequence variants and sensitive quantitative PCR analysis. The combination of this assay principle with a solid-phase format yields a robust assay that is both suitable for large-scale use (21) and easy to set up for detecting single-nucleotide variations for different purposes both in the research laboratory and in the routine clinical laboratory.
References 1. V. A. McKusick, in "Mendelian Inheritance of Man," Vol. 1, 10th ed., p. xxxi. The Johns Hopkins University Press, Baltimore, Maryland, 1992. 2. K. B. Mullis and E A. Faloona, Methods Enzymol. 155, 335 (1987). 3. R. G. H. Cotton, Mutat. Res. 285, 125 (1993). 4. A.-C. Syv~inen and U. Landegren, Hum. Mutat. 3, 172 (1994). 5. A.-C. Syv~inen, K. Aalto-Set~il~i, L. Harju, K. Kontula, and H. S6derlund, Genomics 8, 684 (1990). 6. A.-C. Syv~inen, Clin. Chim. Acta 226, 225, (1994). 7. M. A. Innis and D. H. Gelfand, in "PCR Protocols. A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 3. Academic Press, San Diego, California, 1990. 8. R. Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification" (H. A. Erlich, ed.), p. 35. Stockton Press, New York, 1989. 9. E. S. Kawasaki, in "PCR Protocols. A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 21. Academic Press, San Diego, California, 1990. 10. C. Orrego, in "PCR Protocols: A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds.), p. 451. Academic Press, San Diego, California, 1990. 11. A.-C. Syv~inen, A. Sajantila, and M. Lukka, Am. J. Hum. Genet. 52, 46 (1993). 12. A.-C. Syv~inen and H. S6dedund, Methods Enzymol. 218, 474 (1993). 13. L. Harju, T. Weber, L. Alexsandrova, M. Lukin, M. Ranki, and A. Jalanko, Clin. Chem. 2, 2282 (1993). 14. J. Ihalainen, H. Siitari, S. Laine, A.-C. Syv~inen, and A. Palotie, BioTechniques 16, 938 (1994). 15. A.-C. Syv~en, H. S6dedund, E. Laaksonen, M. Bengtstr6m, M. Turunen, and A. Palotie, Int. J. Cancer50, 713 (1992). 16. A.-C. Syv~en, E. Ikonen, T. Manninen, M. Bengstr6m, H. S6derlund, E Aula, and L. Peltonen, Genomics 12, 590 (1992). 17. A. Suomalainen, E Kollmann, J.-N. Octave, H. S6dedund, and A.-C. Syv~inen, Eur. J. Hum. Genet. 1, 88 (1993). 18. A. Suomalainen, A. Majander, H. Pihko, L. Peltonen, and A.-C. Syv~inen, Hum. Mol. Genet. 2, 525 (1993).
[2] POINTMUTATION DETECTION BY MINISEQUENCING
25
19. V. Juvonen, K. Huoponen, A.-C. Syv~inen, E Aula, E. Nikoskelainen, and M. Savontaus, Hum. Genet. 93, 16 (1994). 20. E. Ikonen, T. Manninen, L. Peltonen, and A.-C. Syv~inen, PCR Methods Appl. 1, 234 (1992). 21. M. Hietala, H. Gr6n, A.-C. Syv~inen, L. Peltonen, and E Aula, Eur. J. Hum. Genet. 1, 296 (1993).