Simultaneous Detection of Multiple Point
`Mutations Using Allele-Specific
`Oligonucleotides
`
`UNIT 9.4
`
`This approach can be used to screen one gene for many allelic mutations or to screen
`several loci for several allelic mutations each. In the basic protocol, pools of radiolabeled
`allele-specific oligonucleotide (ASO) probes are hybridized to dot blots containing
`polymerase chain reaction (PCR)-amplified DNA products generated from one or more
`loci (Fig. 9.4.1). Because tetramethyl ammonium chloride (TMAC) is added to the
`hybridization solution, the melting temperature of each oligonucleotide is independent
`of G-C content and oligonucleotides of the same length can be hybridized simultaneously.
`The pooled probes will give a positive hybridization signal from any PCR-amplified DNA
`sample containing a sequence complementary to any of the ASOs in the pool of
`oligonucleotide sequences. If many PCR-amplified samples are spotted onto a single
`filter, multiple individuals can then be screened simultaneously for many mutant se-
`quences. This multiple ASO hybridization technique is appropriate only for circumstances
`when hybridization with any one of the pooled probes is expected to be uncommon. The
`support protocol describes the removal of radiolabeled probe DNA from the filter in order
`to reuse the filter for further screening. An example highlighted in the Commentary details
`the use of this protocol for studying the cystic fibrosis transmembrane conductance
`regulator (CFTR) gene.
`
`SCREENING PCR-AMPLIFIED DNA WITH MULTIPLE POOLED ASOs
`This approach is particularly powerful when used to screen for rare alleles, but is not
`appropriate for screening commonly occurring alleles. It works well for detecting point
`mutations and small deletions/insertions. PCR-amplified DNA samples that test positive
`are then rescreened individually using single allele-specific oligonucleotides (ASOs) to
`identify which mutation-specific ASO hybridizes. The mutant PCR-amplified DNA can
`also be screened using an ASO corresponding to the normal gene sequence to determine
`whether the individual is heterozygous or homozygous for the mutant allele.
`
`The following steps describe radiolabeling ASOs using T4 polynucleotide kinase, pre-
`paring dot blots of PCR-amplified DNA, and hybridizing pooled radiolabeled ASOs to
`dot blots.
`
`Materials
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
`10× T4 polynucleotide kinase buffer (see recipe)
`200 mM DTT
`10 µM allele-specific oligonucleotide (ASO), prepared just before use by
`diluting 100 µM stock
`10 mCi/ml [γ-32P]dATP (∼3000 Ci/mmol)
`10 U/µl T4 polynucleotide kinase
`25 mM EDTA
`Denaturing solution (see recipe)
`25 ng/µl PCR-amplified products from genes of interest (see Critical Parameters)
`2× SSC (APPENDIX 2)
`TMAC hybridization solution (see recipe)
`TMAC wash solution (see recipe), room temperature and 52°C
`
`Contributed by Barbara Handelin and Anthony P. Shuber
`Current Protocols in Human Genetics (1995) 9.4.1-9.4.8
`Copyright © 2000 by John Wiley & Sons, Inc.
`
`BASIC
`PROTOCOL
`
`Clinical
`Molecular
`Genetics
`
`9.4.1
`
`Supplement 6
`
`GeneDX 1021, pg. 1
`
`

`

`Dot-blot apparatus
`Nylon membrane: e.g., Biotrans+ (ICN Biomedicals) or Biodyne (Pall)
`Whatman 3MM filter paper
`80°C oven
`Sealable bags
`52°C shaking water bath
`X-Omat AR film (Eastman Kodak)
`Additional reagents and equipment for PCR (UNITS 7.1 & 9.3; CPMB UNIT 15.1) and
`preparing dot blots, (CPMB UNIT 2.9B)
`
`CAUTION: 32P and TMAC are hazardous; see APPENDIX 2A for guidelines on handling, storage, and
`disposal.
`
`Label the ASO with 32P
`1. For each ASO to be labeled, prepare the following labeling reaction:
`10 µl H2O
`2 µl 10× T4 polynucleotide kinase buffer
`1 µl 200 mM DTT
`3 µl 10 µM ASO
`3 µl 10 mCi/ml [γ-32P] ATP
`1 µl 10 U/µl polynucleotide kinase.
`Incubate 1 hr at 37°C.
`See Critical Parameters for important tips on design of probes to be used in the labeling
`reaction. The specific probes which are to be used would, of course, be determined by the
`genetic locus of interest.
`2. Stop the reaction with 80 µl of 25 mM EDTA.
`
`Prepare dot blot of PCR-amplified DNA samples
`3. Prepare the dot-blot apparatus according to the manufacturer’s instructions.
`If necessary, clean the dot-blot apparatus because the apparatus must be clean and dry to
`obtain adequate suction.
`4. Estimate the size of membrane that will be necessary to contain the number of samples
`to be analyzed and cut the membrane to the appropriate size to fit the dot-blot
`apparatus. Mark the dry membrane in asymmetric corners so that it can be reoriented
`after hybridization.
`The membrane may be marked with a pen (e.g., Sharpie extra-fine-point marker) or by
`cutting a corner.
`5. Wet the membrane by floating it on water (it should wet immediately), then submerge
`it briefly. Place the membrane on the dot-blot apparatus gasket and assemble the
`manifold—do not apply vacuum until just before loading the samples.
`6. For each sample to be dotted onto the membrane, add 50 µl denaturing solution to a
`1.5-ml microcentrifuge tube. Next, add 8 µl PCR-amplified DNA from the gene of
`interest and mix by vortexing.
`As many as eight PCR-amplified DNA fragments (products) can be combined in a single
`tube and co-dotted onto the membrane (see Critical Parameters). PCR-amplified DNA from
`a multiplex PCR (using 8 (cid:1)l containing 200 ng of each amplification product; see UNIT 9.3)
`can be used.
`
`For each ASO included in the hybridization cocktail (see below), include a PCR-amplified
`DNA from an individual who is a known homozygote or heterozygote for that mutation (as
`a positive control). Include PCR-amplified DNA from an individual who does not have the
`mutation and a “no-DNA” PCR sample as negative controls.
`
`Current Protocols in Human Genetics
`
`Simultaneous
`Detection of
`Multiple Point
`Mutations Using
`Allele-Specific
`Oligonucleotides
`
`9.4.2
`
`Supplement 6
`
`GeneDX 1021, pg. 2
`
`

`

`ASO position
`
`R117H 621+1
`
`508
`507
`
`G542X
`1717-1
`
`S549N
`R553X
`G551D
`
`R560T
`
`W1282X N1303K
`
`exon #
`
`4
`
`10
`
`11
`20
`carry out PCR amplification
`on CFTR gene
`
`21
`
`prepare four replicate dot-blots
`of amplified DNA
`
`hybridize with
` 508 probe
`
`hybridize with
`pool of 5
`mutations
`
`hybridize with
`pool of 6
`mutations
`
`hybridize with
`N( 508)
`
`1 2 3 4 5 6 7 8 9101112
`
`←
`
`←
`
`ABCDE
`
`N( 508)
`
`1 2 3 4 5 6 7 8 9 10 1112
`
`←
`
`←
`
`ABCDE
`
`Pool 2
`
`perform independent hybridization
`for mutation specification
`
`A (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`B
`
`A (+)
`B
`
`Pool 2
`probes
`1 2 3 4 5
`
`6
`
`∆507
`
`R117H
`
`←←
`
`←←
`
`621+1
`
`S549N
`
`R560T
`
`1717-1
`
`1 2 3 4 5 6 7 8 9 10 1112
`
`←
`
`←
`
`Pool 1
`probes
`1 2 3 4 5
`
`ABCDE
`
`Pool 1
`
`1 2 3 4 5 6 7 8 9 10 1112
`
`←
`
`←
`
`←
`
`←
`
`ABCDE
`
`508
`
`A (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`
`B A
`
` (+)
`B
`
`G542X
`
`G551D
`
`R553X
`
`←←
`
`W1282X
`
`N1303K
`
`Figure 9.4.1 Detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using
`allele-specific oligonucleotides.
`
`7. When all samples have been added to denaturing solution, apply the vacuum to the
`dot-blot apparatus. Add the entire volume of each sample to a well, avoiding bubbles
`on the filter. Prepare Whatman 3MM filter paper saturated with 2× SSC.
`A common mistake at this point is to place a sample in the wrong well. Use bromphenol
`blue in the denaturing solution to keep track of sample loading.
`
`Clinical
`Molecular
`Genetics
`
`9.4.3
`
`Current Protocols in Human Genetics
`
`GeneDX 1021, pg. 3
`
`

`

`8. With the vacuum still on, remove the upper block of the apparatus, quickly remove
`the membrane, and place it onto Whatman 3MM filter paper saturated with 2× SSC.
`Let sit for 2 min.
`9. Fix the DNA onto the membrane by placing it for 15 min in an 80°C oven. Rewet the
`membrane in water and transfer it to a sealable bag for hybridization.
`
`The filters may be temporarily stored by keeping in a dark place, at room temperature.
`
`Hybridize 32P-labeled ASOs to dot blots and autoradiograph
`10. Add an appropriate amount of hybridization solution containing the 32P-labeled ASO
`pooled probes to each bag. Incubate 2 hr to overnight, with shaking, in 52°C water
`bath.
`
`For example, for a standard 96-well format membrane, use 10 ml of hybridization solution.
`The optimal concentration of each ASO probe should be determined empirically, but should
`be in the range of 0.03 to 0.15 pmol/ml of hybridization solution. To reduce background
`hybridization, add ≥20-fold excess unlabeled ASO corresponding to the “normal” allele.
`
`This protocol has been optimized for 17-mer ASO probes.
`
`11. Remove the membrane from the bag and wash with vigorous agitation as follows: 20
`min in 200 ml TMAC wash solution, room temperature, followed by 20 min in 300
`ml TMAC wash solution, 52°C.
`12. Blot membranes on Whatman 3MM filter paper to dry and expose to X-Omat AR
`film 1 hr to overnight at −70°C.
`
`SUPPORT
`PROTOCOL
`
`STRIPPING OLD PROBES AND REHYBRIDIZATION
`Filters can be stripped of previously hybridized probe and rehybridized several times,
`depending on the amount of DNA (control and experimental) on the filters. If the
`signal-to-noise ratio allows reading of the appropriate hybridization signals from positive-
`and negative-control DNA, and the amount of experimental and control DNA on the filter
`is equivalent, then the experimental results can be interpreted.
`
`Additional Materials
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
`Hybridized membrane (first basic protocol)
`TMAC wash solution (see recipe), freshly prepared and prewarmed to 15°C
`above previous hybridization temperature
`Agitating water bath, 15°C above previous hybridization temperature
`
`CAUTION: Radiolabeled hybridized membranes and TMAC are hazardous; see APPENDIX 2A for
`guidelines on handling, storage, and disposal.
`
`1. Add previously hybridized membrane to 300 ml prewarmed TMAC wash solution in
`a washing dish. Wash 1 hr with vigorous agitation in a water bath at 15°C above
`previous hybridization temperature.
`
`2. Remove filter from the wash and blot dry on Whatman 3MM filter paper.
`
`3. Autoradiograph to confirm that the previously hybridized probe has been removed.
`
`4. Hybridize filters as described in steps 10 to 12 of the basic protocol, or store dry at
`room temperature (they can be stored indefinitely in resealable bags).
`
`Current Protocols in Human Genetics
`
`Simultaneous
`Detection of
`Multiple Point
`Mutations Using
`Allele-Specific
`Oligonucleotides
`
`9.4.4
`
`GeneDX 1021, pg. 4
`
`

`

`REAGENTS AND SOLUTIONS
`
`Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
`APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
`
`Denaturing solution
`500 mM NaOH
`2.0 M NaCl
`25 mM EDTA (prepare freshly)
`0.0001% (w/v) bromphenol blue (add just before use)
`Prepare fresh
`T4 polynucleotide kinase buffer, 10×
`700 mM Tris⋅Cl, pH 7.6
`100 mM MgCl2
`50 mM spermidine
`Store frozen
`Tetramethylammonium chloride (TMAC) solution
`Dissolve 657.6 g TMAC (mol. wt. = 109.6) in 1 liter H2O. Filter solution through
`Whatman no. 1 filter paper and determine the precise concentration by measuring
`the refractive index (n) of a three-fold diluted solution. The molarity (M) of the
`diluted solution = 53.6 (n − 1.331) and the molarity of the stock solution = 3 × M.
`Store TMAC at room temperature in brown bottles.
`CAUTION: TMAC is hazardous; see APPENDIX 2A for guidelines on handling, storage, and
`disposal.
`TMAC hybridization solution
`6 ml TMAC solution (see recipe; ∼3 M final)
`600 µl 10% (w/v) SDS (0.6% final)
`20 µl 0.5 M EDTA (1 mM) final
`1 ml 0.1 M Na3PO4, pH 6.8 (10 mM final)
`1 ml 50× Denhardt solution (APPENDIX 2; 5× final)
`40 µl 10 mg/nl yeast RNA (40 µg/ml final)
`1.34 ml H2O
`CAUTION: TMAC is hazardous; see APPENDIX 2A for safety guidelines.
`TMAC wash solution
`60 ml TMAC solution (see recipe; ∼3 M final)
`200 µl 0.5 M EDTA (1 mM final)
`10 ml 0.1 M Na3PO4, pH 6.8 (10 mM final)
`6 ml 10% (w/v) SDS (0.6% final)
`23.8 ml H2O
`CAUTION: TMAC is hazardous; see APPENDIX 2A for safety guidelines.
`
`COMMENTARY
`Background Information
`Review of current human molecular genetics
`literature reveals that the paradigm of sickle cell
`anemia, in which one mutation accounts for
`essentially 100% of the cases of sickle cell
`phenotype, is the exception rather than the rule.
`As associations between genes and disease phe-
`notypes are discovered and the mutations that
`confer the disease phenotype are determined, a
`
`new rule is rapidly being established: DNA
`diagnosis of genetic diseases typically requires
`analysis of many alternative mutations, each of
`which confers the disease phenotype.
`Mutation analysis using ASO probes is di-
`rect and is less subject to errors or misinterpre-
`tation than other techniques for detecting spe-
`cific sequences; i.e., if specific hybridization of
`the probe is observed, the complementary se-
`
`Current Protocols in Human Genetics
`
`Clinical
`Molecular
`Genetics
`
`9.4.5
`
`GeneDX 1021, pg. 5
`
`

`

`quence is present. However, in order to analyze
`multiple ASOs, it is usually necessary to per-
`form separate hybridizations and washes for
`each probe. This is because pooling of ASOs in
`the same hybridization would generally require
`that probes be designed with identical melting
`temperatures (Tm) so that the stringency of
`hybridization can be controlled to reduce non-
`specific binding. In a pool of three or four
`ASOs, satisfactory results can be obtained by
`designing probes of different lengths or by
`adjusting placement of the ASO at the target
`sequence. However, as the pool gets larger, this
`approach becomes progressively more com-
`plex.
`The use of the quaternary ammonium salt
`TMAC as an adjunct in hybridization reactions
`essentially eliminates the dependence of Tm on
`G-C content (see CPMB UNITS 2.9 & 6.4; and Mel-
`chior and von Hippel, 1973). TMAC acts
`through a nonspecific salt effect to reduce hy-
`drogen-bonding (H-bond) energies between G-
`C base pairs (Marky et al., 1988). At the same
`time, it binds specifically to A-T base pairs and
`increases the thermal stability of the H-bonds
`between A-T base pairs. At 3 M TMAC, these
`opposing influences effectively reduce the dif-
`ference in bonding energy between the triple-
`H-bond G-C base pair and the double-H-bond
`A-T base pair. The net result is that the Tm of a
`probe in 3 M TMAC is a function of probe
`length alone. Thus, if all probes in an ASO pool
`are the same length, their Tm will be virtually
`identical.
`A related consequence of eliminating the
`bonding energy differences between A-T and
`G-C base pairs is that the slope of the melting
`curve for each probe increases. Together the
`effects of TMAC in hybridization allow the
`stringency of hybridization to increase to the
`point that single-base differences can be re-
`solved and nonspecific hybridization minimized.
`Specificity of hybridization is especially im-
`portant when multiple ASO probes with the
`same, or closely overlapping, targets are pooled;
`as, for example, in analyzing mutations S549N,
`G551D, and R553X in the CFTR gene where
`these mutations have identical target locations
`and differ in sequence by only 1 bp (Fig. 9.4.1).
`Thus, ASOs for mutations occurring in close
`proximity on the same target sequence do not
`cross-hybridize under TMAC hybridization con-
`ditions, as they tend to do with the allele-spe-
`cific priming (ASP) technique (Ferrie et al.,
`1992). Equally important, TMAC hybridiza-
`tion works with a heterogeneous population of
`
`target DNA (i.e., multiple PCR-amplified DNAs
`co-dotted on a membrane) without generating
`nonspecific background hybridization (Shuber
`et al., 1992).
`The method is appropriate only when hy-
`bridization with any one of the ASOs in a pool
`is expected to be somewhat uncommon. For
`example, this method would not be useful to
`genotype the common human histocompatibil-
`ity leukocyte antigen (HLA) alleles as almost
`every sample would have a positive signal when
`hybridized with ASO pools containing any of
`the most common HLA alleles. However, for
`rare disease-causing alleles at various genetic
`loci, this approach is appropriate.
`
`Critical Parameters
`This method should be robust for both speci-
`ficity and sensitivity. That is, it should be pos-
`sible to analyze a complex sample (i.e., one
`containing multiple PCR products from one or
`several loci) with multiple ASOs. One remark-
`able feature of the TMAC hybridization method
`is that it is possible to hybridize simultaneously
`several ASO probes that overlap significantly
`at their target sequence. ASO probes may differ
`by only a single base without reducing specific
`signal via competitive hybridization.
`A significant advantage in using this proce-
`dure is that designing ASO probes only in-
`volves the necessity to observe two parameters:
`(1) all ASO probes in a single pool should be
`the same length (the basic protocol is optimized
`for a 17-base ASO probe) and (2) the position
`of the discriminating single-base mismatch be-
`tween the oligonucleotide probe and target se-
`quence should not be located at the end of the
`ASO. The basic protocol is based on the single-
`base mismatch being located 5 to 7 bases from
`the 5′ end of the ASO probe.
`Hybridization using pools of ≤20 ASO
`probes yields acceptable results but the upper
`limit to the number of ASO probes that can be
`combined is probably >20. ASOs of the same
`length, but with differing G-C content, can be
`pooled because TMAC hybridization elimi-
`nates dependence of the Tm of the probe on the
`G-C content.
`Pools of PCR-amplified DNA from several
`genes of interest can be co-dotted onto a filter
`and screened simultaneously. The PCR-ampli-
`fied DNA samples can be pooled from separate
`PCR amplification reactions (UNIT 7.1; CPMB UNIT
`15.1) or prepared in a multiplex PCR-amplifica-
`tion reaction (UNIT 9.3).
`
`Current Protocols in Human Genetics
`
`Simultaneous
`Detection of
`Multiple Point
`Mutations Using
`Allele-Specific
`Oligonucleotides
`
`9.4.6
`
`GeneDX 1021, pg. 6
`
`

`

`Table 9.4.1 Troubleshooting Guide for Mutation Analysis Using Allele-Specific Oligonucleotides Probes
`
`Problem
`
`Possible cause
`
`Solution
`
`Hybridization signal from
`sample is low or absent
`
`Specific activity of labeled ASO probe
`is low
`Probe is partially hydrolyzed or
`degraded
`No denaturing solution added to
`PCR-amplified DNA sample
`
`Probe not added to the hybridization
`cocktail
`No DNA spotted onto membrane
`
`Background signal is high
`
`Hybridization and wash temperature are
`not high enough
`
`Labeled probe remains on membrane
`after the washes have been done
`Agitation of washes is too slow
`
`Inadequate volume of wash solution
`
`Repeat the labeling procedure with
`fresh ASO and label
`Prepare new probe
`
`Denature, spot, and hybridize a
`second aliquot from amplified
`sample
`Add probe to hybridization solution
`and proceed as usual
`Repeat denaturation, spotting, and
`hybridizations with new aliquot of
`amplified sample
`
`Do not allow membrane to
`completely dry out; return damp
`membrane to a new wash solution
`and repeat washes at given
`temperature; omit room temperature
`wash
`Perform an additional 5-10 min wash
`at the required wash temperature
`Repeat washes with increased
`agitation; check to be sure that
`membrane has not become stuck to
`side of wash container
`Add additional wash solution and
`repeat
`
`Troubleshooting
`The problems encountered are one of two
`types: the specific hybridization signal is too
`low or the background hybridization is too high.
`Approaches to troubleshooting these problems
`are summarized in Table 9.4.1.
`
`Anticipated Results
`DNA samples that contain any sequence
`complementary to one of the ASO probes in a
`pool will give a positive hybridization signal.
`Of course, it is not possible to determine which
`of the pooled probes is responsible for a positive
`signal. Positive DNA samples are reanalyzed
`singly with individual ASO probes to determine
`which of the pooled ASOs hybridized. A posi-
`tive result does not discriminate between ho-
`mozygosity and heterozygosity. Thus, a posi-
`tive sample must be rehybridized with the
`ASOs corresponding normal sequence to de-
`termine if the individual has one or two copies
`of the mutant sequence.
`
`Time Considerations
`PCR amplification of the gene of interest
`and the entire basic protocol can be performed
`in a single day if the hybridization conditions
`are optimized for the pool of ASO probes being
`used, allowing a 2-hr hybridization. The hy-
`bridization time can be as long as overnight to
`62 hr for scheduling convenience. The length
`of time allowed for posthybridization washes
`is minimal, as are the number of washes. Unlike
`the washes used for hybridizations using SSC,
`the number and duration of washes are mini-
`mal, but critically timed.
`
`Example: Detection of Mutations
`in the CFTR Genes Using ASOs
`Consider the detection of mutations in the
`cystic fibrosis transmembrane conductance
`regulator (CFTR) gene in a clinical laboratory.
`Diagnosis of 12 CFTR mutations would pro-
`ceed as follows (and as outlined in Figure
`9.4.1): Samples are amplified in a multiplex
`
`Current Protocols in Human Genetics
`
`Clinical
`Molecular
`Genetics
`
`9.4.7
`
`GeneDX 1021, pg. 7
`
`

`

`reaction to generate PCR products from five
`exons in the CFTR gene (Shuber et al., 1992).
`The PCR-amplified DNA is then dotted onto a
`96-well dot-blot apparatus (80 experimental
`and 16 control samples are analyzed simulta-
`neously). Four replicate dot-blot membranes
`are generated; one for each ASO pool to be
`hybridized. Replicate membranes are then hy-
`bridized with a cocktail of radiolabeled ASO
`probes. The composition of each pool is deter-
`mined by the relative frequency of specific
`mutations in the population being analyzed.
`More common mutations are not pooled (∆508
`probe). ASOs that recognize rare mutant allele
`are similarly pooled (5- and 6-mutation pools).
`The fourth replicate is hybridized to a probe
`containing the normal sequence at the ∆508
`position.
`Autoradiograms of these four hybridizations
`are analyzed as follows: In the ∆F508 mem-
`brane, the ∆F508 homozygote is read in column
`6, rows D and E (duplicate spots; see arrows).
`In the Pool 1 membrane, the sample in column
`4, rows D and E is read as the Pool 1 positive
`(unknown heterozygote or homozygote; see
`arrows). An example of a compound heterozy-
`gote can be found in column 5, rows D and E,
`on the ∆F508 and Pool 2 membranes.
`DNA samples that give a positive signal are
`rescreened using individual ASO probes in the
`hybridizations to determine which ASO probe
`in the pool hybridized to the sample DNA. Note
`in Fig. 9.4.1 that the number of samples giving
`a positive hybridization signal is small, espe-
`cially for the ASO pool containing the rarest
`mutant alleles. In this figure, columns 1 to 5 and
`1 to 6 are generated by aligning filters. The rows
`labeled A(+) contain positive controls. The rows
`labeled B are experimental sample rows.
`Although not shown in Fig. 9.4.1, positive
`samples also are hybridized with an ASO that
`recognizes the sequence of the normal allele to
`discriminate between a heterozygous and ho-
`mozygous genotype (e.g., G551D/G551D or
`
`G551D/normal). This step may not always be
`necessary, (e.g., in a phenotypically normal
`individual who is suspected only of being a
`carrier/heterozygote).
`
`Literature Cited
`Ferrie, R.M., Schwarz, M.J., Robertson, N.H., Vau-
`din, S., Super, M., Malone, G., and Little, S.
`1992. Development, multiplexing, and applica-
`tion of ARMS tests for common mutations in the
`CFTR gene. Am. J. Hum. Genet. 51:251-262.
`Marky, L.A., Blumenfeld, K.S., and Breslauer, K.J.
`1988. Differential effect of tetramethylam-
`monium chloride and sodium chloride on duplex
`melting temperature of deoxyoligonucleotides:
`Resolution of a salt effect into specific and non-
`specific components. Can. J. Chem. 66:836-838.
`Melchior, W.B. and von Hippel, P.H. 1973. Altera-
`tion of the relative stability of dA-dT and dG-dC
`base pairs in DNA. Proc. Nat. Acad. Sci. U.S.A.
`70:298-302.
`Shuber, A.P., Skoletsky, J., Stern, R., and Handelin,
`B.L. 1992. Efficient 12-mutation testing in the
`CFTR gene: A general model for complex muta-
`tion analysis. Hum. Molec. Genet. 2:159-163.
`
`Key Reference
`Shuber et al., 1992. See above.
`Original description and validation of the method.
`
`Wood, W.I., Gitschier, J., Lasky, L.A., and Lawn,
`R.M. 1985. Base composition-independent hy-
`bridization in tetramethylammonium chloride: A
`method for oligonucleotide screening of highly
`complex gene libraries. Proc. Natl. Acad. Sci.
`U.S.A. 82:1585-1588.
`Establishes the empirical conditions for use of
`TMAC in hybridizing degenerate oligonucleotide
`pools to cDNA library clones. This is an important
`application of TMAC conditions and is the closest
`to the application described here.
`
`Contributed by Barbara Handelin
` and Anthony P. Shuber
`Integrated Genetics
`Framingham, Massachusetts
`
`Simultaneous
`Detection of
`Multiple Point
`Mutations Using
`Allele-Specific
`Oligonucleotides
`
`9.4.8
`
`Current Protocols in Human Genetics
`
`GeneDX 1021, pg. 8
`
`