This Sample Unit contains the full text of the published Unit, including expert commentary sections with critical information designed to ensure the success of your experiments.

UNIT 7.1

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Amplification of Sequences from Affected Individuals

Contributed by Hugh C. Watkins
Brigham and Women's Hospital and Harvard Medical School
Boston, Massachusetts

This unit describes methods for obtaining DNA sequences from an individual affected by a genetic disorder. Target sequences that may be present at very low copy number in patient samples are amplified by the polymerase chain reaction (PCR). These sequences can then be examined for disease-causing mutations using protocols for the detection of point mutations or small deletions or insertions (UNITS 7.2-7.6). Depending upon the circumstances, the desired sequences may be those of the coding sequence (cDNA or individual exons) or of genomic DNA. In many instances, it is desirable to confine analysis to the coding sequence and the splice-donor and splice-acceptor sites in the immediate flanking intron sequences, where disease-causing mutations are most likely to be found.

Amplification protocols are based on the PCR and therefore require knowledge of the normal sequence of the candidate gene to design oligonucleotide primers. If only the cDNA sequence is known, analyses in affected individuals depend on obtaining mRNA molecules. The first basic protocol describes harvesting mRNA from peripheral lymphocytes, which can even be utilized for genes with tissue-specific patterns of expression. This technique has the advantage of ease of access to appropriate patient samples and also may provide a more efficient means of screening coding sequences than would analysis of individual exons. An alternate protocol describes modifications in PCR conditions that facilitate mutation analysis and sequencing. When the genomic sequence for a given candidate gene is known, the second basic protocol may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.

NOTE: Experiments involving PCR and RNA require extremely careful technique to prevent contamination and RNA degradation, see APPENDIX 2D.

STRATEGIC PLANNING

Amplification from cDNA is based on the use of nested oligonucleotide primers for two successive rounds of PCR (Fig. 7.1.1). An outer reverse primer (R) is selected for reverse transcription--e.g., this primer may be situated in the terminal 3' untranslated sequence. The same primer is used for the first-round amplification, together with an outer forward primer (F). A first-round product of ~1 kb is usually appropriate. Pairs of inner primers (f' and r'; f'' and r'') are designed to amplify sections of coding sequence appropriate for the intended method of screening for mutations (see Commentary for details on this and the specific design of individual oligonucleotides). Primers should be positioned so as to provide sufficient overlap for detection of mutations near the end of each segment, because point mutations within primer sequences are typically not detectable after PCR.

Amplification of genomic DNA sequences is achieved using a single primer pair for each target segment. Primers may be designed to amplify one exon at a time, as long as the size of the exon is compatible with the screening device that is to be used. It may be desirable for primers to be situated with their 3' ends close to the exon/intron boundary to minimize inclusion of intronic sequences, which are more likely to contain noncoding polymorphisms.

BASIC PROTOCOL 1

AMPLIFICATION OF RNA FROM LYMPHOCYTES

This protocol describes the isolation of total RNA from peripheral blood lymphocytes, reverse transcription into cDNA, and nested PCR amplification of cDNA sequences from a desired gene.

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.

Transformed B cells from saturated Epstein-Barr virus (EBV) cultures (APPENDIX 3H) or nontransformed lymphocytes isolated by Ficoll-Hypaque gradient centrifugation (UNIT 10.4)

PBS (APPENDIX 2), ice cold

Diethylpyrocarbonate (DEPC)-treated H₂O (see recipe)

1.25 mM 4dNTP mix (APPENDIX 2)

10× PCR amplification buffer (APPENDIX 2) containing 15 mM MgCl₂

25-mer oligonucleotide primers (Fig. 7.1.1): outer (reverse and forward) and inner (reverse and forward)

RNasin (Promega)

Reverse transcriptase [e.g., Moloney murine leukemia virus (MoMuLV)-RT]

Taq DNA polymerase (Perkin-Elmer Cetus)

Mineral oil

Sieving agarose (e.g., Nusieve, FMC Bioproducts)

DNA molecular size markers

1.5- and 0.5-ml polypropylene microcentrifuge tubes, clean and RNase-free

Thermal cycler

42°C water bath

Beckman JS-4.2 rotor or equivalent

Additional reagents and equipment for isolation of RNA by single-step guanidinium method (UNIT 10.4) and agarose gel electrophoresis (UNIT 2.7)

CAUTION: Human lymphocytes, Epstein-Barr virus, and DEPC are hazardous; see APPENDIX 2A for guidelines on handling, storage, and disposal.

NOTE: All water and salt solutions (except Tris) should be freshly treated with DEPC. Use sterile, disposable plasticware where possible. See CPMB UNIT 4.1 for guidelines on standard methods to protect against contaminating RNases.

Isolate the lymphocytes

Collect transformed B cells from 30 ml of saturated EBV culture or nontransformed lymphocytes extracted by Ficoll-Hypaque gradient centrifugation from 20 to 30 ml fresh whole blood.

The yield from these amounts is ~10⁹cells. Transformed or nontransformed cells appear to work equally well, but the availability of a transformed line is a great advantage should analysis of repeat samples be necessary. Cells frozen in DMSO and stored at 70°C are adequate and are stable for at least 1 year.

Wash cells once with ice-cold PBS. Centrifuge 5 min at 300 g (1000 rpm in Beckman JS-4.2 rotor), 4°C.

Extract total RNA

Extract total RNA from the cells by the single-step guanidinium thiocyanate method.

A commercial kit (e.g., RNAzol, Biotecx Laboratories) may also be used. Either method permits recovery of total RNA from small numbers of cells and, because ultracentrifugation is not required, many samples can be processed in parallel.

Resuspend RNA in 100 µl DEPC-treated water in microcentrifuge tubes. Store in two 50-µl aliquots at 70°C.

Expect the RNA concentration to be ~1 µg/ml (100 µg total). Rough quantitation can be provided by spotting a 1-µl aliquot on an ethidium bromide-stained agarose gel for comparison with known amounts of tRNA. RNA stored at 70°C is stable for 1 year, but will tend to be degraded by multiple freeze-thawing. If this is anticipated, divide into aliquots.

Reverse transcribe RNA into cDNA

Set up the following 20-µl reaction in a 0.5-ml microcentrifuge tube:

2 µl 1.25 mM 4dNTP mix

2 µl 10× PCR amplification buffer

500 ng reverse (antisense) outer primer (60 pmol)

20 U RNasin

2 µg (~2 µl) RNA (from step 4)

50 U reverse transcriptase

DEPC-treated H₂O to 20 µl.

Incubate 45 min at 42°C. Heat-inactivate the reverse transcriptase 10 min at 90°C. After cooling, microcentrifuge briefly at top speed.

Control reactions (identical except for absence of RNA) should be interspersed with test samples and processed in parallel.

The cDNA reaction product from this first-strand synthesis forms the substrate for the first PCR amplification (step 6); PCR buffer and sufficient reverse primer are included to facilitate the amplification reaction. The cDNA product is stable and can be stored overnight at 4°C until amplified, or indefinitely at 20°C.

Amplify the cDNA

Set up the following 100-µl reaction in a 0.5-ml microcentrifuge tube:

16 µl 1.25 mM 4dNTP mix

8 µl 10 PCR amplification buffer

500 ng forward (sense) outer primer (60 pmol)

20 µl cDNA (step 5)

DEPC-treated H₂O to 100 µl.

Add 2.5 U Taq DNA polymerase. Mix, microcentrifuge briefly at top speed, and overlay with 2 drops of mineral oil if appropriate for thermal cycler model.

To amplify multiple samples, prepare a cocktail of the reaction components (including the polymerase) and add 80 µl directly to 20 µl of each cDNA.

Carry out PCR using the following amplification cycles:

40 cycles:	30 sec	94°C	(denaturation)
	1 min	55°C	(annealing)
	2 min	72°C	(extension)
Final step:	indefinitely	4°C	(hold).

Forty cycles are necessary due to the low initial copy number of target sequences; long extension times are recommended because the outer primer pair product will generally be ~1 kb in length. The 4°C holding step ensures product stability until reactions are removed from the thermal cycler. After first-round amplification, insufficient specific product is present to allow confirmation by gel electrophoresis.

Perform second nested-PCR amplification

Dilute 10 µl first-round PCR product in 1 ml water. Use 5 µl of this 1:100 dilution as the template for the second PCR amplification.

Both the first PCR product and the dilution should be saved and are stable indefinitely at 4°C. The dilution (1:1000 final) effectively removes residual outer primers and dilutes the nonspecific products of the first-round PCR, reducing the likelihood of their amplification in the second round.

For each sample, set up the following 50-µl reaction in a 0.5-ml microcentrifuge tube:

8 µl 1.25 mM 4dNTP mix

5 µl 10 PCR amplification buffer

250 ng each forward and reverse inner primers (30 pmol)

5 µl 1:100 dilution of first-round PCR product (from step 8)

H₂O to 50 µl.

Add 1.25 U Taq DNA polymerase. Mix, microcentrifuge briefly at top speed, and overlay with mineral oil if appropriate for thermal cycler model.

Further control reactions (identical except for absence of first amplification product) should be interspersed with test samples and processed in parallel.

To amplify multiple samples, a cocktail of the reaction components (including the polymerase) is prepared and 45-µl aliquots are added to each of a new set of microcentrifuge tubes containing the 5 µl of the first PCR product.

Carry out PCR using the following amplification cycles:

30-40 cycles:	30 sec	94°C	(denaturation)
	1 min	55°C	(annealing)
	1 min	72°C	(extension)
Final step:	indefinitely	4°C	(hold).

Thirty cycles are generally sufficient to produce adequate product; this can be confirmed by electrophoresis. The optimum cycle times and temperatures depend on the specific primer and gene sequences, the length of product, and the thermal cycler model (CPMB UNIT 15.1).

Check results by agarose gel electrophoresis

Electrophorese 10 µl from each second-round reaction on a sieving agarose gel stained with ethidium bromide. Include a lane that contains a known quantity of molecular size markers.

For PCR products between 100 and 1000 bp, a 3% Nusieve (FMC Bioproducts) gel is appropriate. A clean product band of appropriate size should be readily visible in all samples except the blanks. Note the presence of any aberrant bands (see Commentary) and keep a photograph of the gel.

ALTERNATE PROTOCOL

MODIFIED SECOND-ROUND AMPLIFICATION OF cDNA

The second-round product obtained in step 10 of the basic protocol is appropriate for applications such as mutation analysis by RNase protection (UNIT 7.2) or denaturing gradient gel electrophoresis (DGGE; UNIT 7.5). For other applications, however, modifications to the second-round amplification may be necessary; these can be implemented after successful amplification using the basic procedure is confirmed (step 11).

Applications such as heteroduplex analysis (UNIT 7.3) or chemical cleavage (UNIT 7.6) may require 5'-³²P-labeled product. For these applications, the second-round PCR is performed as described, but using an end-labeled primer. The 5' ends of either or both primers are labeled prior to use in the PCR reaction by the action of T4 polynucleotide kinase in the presence of [-³²P]ATP (APPENDIX 3E).

Alternatively, the second PCR can be performed using primers containing restriction enzyme sites to facilitate subcloning of the final product, e.g., for sequencing. The enzyme sites selected must not be present in the target DNA sequence and should generate cohesive ends that will facilitate cloning into the polylinker of the chosen vector. Two measures are necessary to ensure efficient restriction of the PCR product. First, the oligonucleotide primers should have a "cap" of three nucleotide residues 5' to the enzyme site sequence (which is itself 5' to the target DNA sequence). Second, because of the tendency of the PCR reaction to produce product with incomplete ends, the second-round PCR product should be filled in by treatment with Klenow fragment to ensure that the complete enzyme site is generated.

BASIC PROTOCOL 2

AMPLIFICATION OF GENOMIC DNA

When the genomic sequence of the candidate gene is available, amplified coding sequences can easily be obtained by PCR amplification of individual exons. This may be preferable to nested-PCR amplification of cDNA if the gene contains few exons or if known mutations in specific exons are to be identified. Genomic DNA amplification is also appropriate for analysis of noncoding regions, such as introns or 5' regulatory sequences. Because the initial copy number will be higher than for amplification of cDNA, a single round of PCR is adequate.

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.

Genomic DNA (APPENDIX 3B)

1.25 mM 4dNTP mix (APPENDIX 2)

10× PCR amplification buffer ( APPENDIX 2) containing 15 mM MgCl₂

Oligonucleotide primers: reverse and forward

Taq DNA polymerase (Perkin-Elmer Cetus)

Mineral oil

Sieving agarose (e.g., Nusieve, FMC Bioproducts)

DNA molecular size markers

1.5- and 0.5-ml polypropylene microcentrifuge tubes

Thermal cycler

Additional reagents for agarose gel electrophoresis (UNIT 2.7)

Dilute each genomic DNA sample to be analyzed to 100 ng/µl and heat-denature 5 min at 95°C.

Heat denaturation removes any residual contaminating nuclease activity and facilitates denaturation in the first PCR cycle (an extended first denaturation step is therefore unnecessary). Store DNA samples indefinitely at 4°C. Samples are ready for use without further heat denaturation.

For each sample, set up the following 50-µ l reaction in 0.5-ml microcentrifuge tubes:

8 µl 1.25 mM 4dNTP mix

5 µl 10 PCR amplification buffer

250 ng each reverse and forward primers

200 ng genomic DNA (2 µl from step 1)

Sterile H₂O to 50 µl.

Add 1.25 U Taq DNA polymerase. Mix, microcentrifuge briefly at top speed, and overlay with mineral oil if appropriate for thermal cycler model.

Control reactions (identical to patient sample reactions except for absence of DNA) should be interspersed and processed in parallel.

For multiple samples, prepare a cocktail of all the reaction components (including the polymerase) and aliquot 48 µl into each microcentrifuge tube containing 2 µl of the sample DNA. Reaction volumes can be increased by scaling up volume of all reagents if multiple analyses of the amplified sequence are anticipated.

Carry out PCR using the following amplification cycles:

30 cycles:	30 sec	94°C	(denaturation)
	1 min	55°C	(annealing)
	1 min	72°C	(extension)
Final step:	indefinitely	4°C	(hold).

Electrophorese 10 µl from each second-round reaction on a sieving agarose gel stained with ethidium bromide. Include a lane that contains a known quantity of molecular size standard.

For PCR products between 100 and 1000 bp a 3% Nusieve gel is appropriate. A clean product band of appropriate size should be readily visible in all samples except the blanks.

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.

Diethylpyrocarbonate (DEPC)-treated water

Add 0.2 ml DEPC to 100 ml sterile H₂O; shake vigorously. Autoclave to inactivate residual DEPC. Prepare fresh before use.

CAUTION: DEPC is hazardous; see APPENDIX 2A for guidelines on handling, storage, and disposal.

COMMENTARY

Background Information

Amplification of candidate gene cDNA from RNA of lymphocytes depends upon the phenomenon of ectopic, or promiscuous, gene expression. Tissue-specific genes--not generally expressed in lymphocytes-are in fact transcribed at very low levels in most, and probably all, tissues (Chelly et al., 1988; Sarkar and Sommer, 1989). A basal level of expression exists, presumably because suppression of transcription is not complete, with copies of the transcript present at very much less than one copy per cell. The amplification provided by two successive rounds of PCR can provide sufficient product even when starting from a few copies of mRNA. Nested primers provide sufficient specificity to amplify only the target sequences. cDNA sequences obtained in this way have been used successfully to detect disease-causing mutations in human genes without the need for obtaining affected tissues (Roberts et al., 1990; Rosenzweig et al., 1991; Watkins et al., 1992).

Critical Parameters and Troubleshooting

RNA integrity

Degradation by contaminating RNase enzymes will prevent successful amplification. Partial degradation of RNA can result from preparation from lysed cells or repeated freeze-thawing of the RNA sample and can produce aberrant (not full-length) amplification products. These products can appear as artifacts on gels (see below). RNA should be stored in multiple aliquots and those used for each experiment should be recorded.

Difficulties with reverse transcription

In general, better results are obtained with a specific reverse primer than with an oligo(dT) primer, as specificity is maximized from the start. Problems may be encountered with reverse transcription in regions of stable secondary structure of the mRNA molecule (GC-rich regions). Such problems may be dealt with by amplifying shorter regions of cDNA or by using alternative enzymes that operate at higher temperatures (e.g., GeneAmp thermostable rTth reverse transcriptase from Perkin-Elmer Cetus), thereby allowing unfolding of the RNA template. Reverse transcriptase activity is described in CPMB UNIT 3.7.

Strategy for dividing the cDNA molecule

The initial amplification efficiency depends on the production of full-length product by reverse transcription. In practice, a first-round product of ~1 kb is usually appropriate. The chosen method of screening for mutations--e.g., by RNase protection (UNIT 7.2) or single-strand conformation polymorphism (SSCP; UNIT 7.4)--will determine the optimum length of second-round product (e.g., 500 bp for RNase protection, but shorter fragments, 250 bp, for SSCP). In this way, a cDNA sequence can be divided into overlapping sections of ~1 kb, each of which is the template for two or more nested second-round amplifications. Primers should be positioned to provide sufficient overlap for detection of mutations near the end of a sequence.

Oligonucleotide primer selection

Specific oligonucleotide pairs must be designed with the usual considerations such as G-C content and potential secondary structure. Primers consisting of 25-mers are adequate; longer primers are unlikely to confer greater specificity. When possible, primers should be placed in regions of sequence divergent from other related genes (e.g., in 3' untranslated sequences). It is also valuable to bracket regions of known introns or features that provide specific identification of the desired gene sequence. Computer programs may assist in the design of optimal primers but constraints on primer position limit their usefulness. Most primers work without problem; a few will not work regardless of PCR conditions and need to be remade for an adjacent site.

Confirmation of cDNA product

If the region amplified contains an intron, discrimination between amplified RNA and DNA is simple based on size alone. With first-round primers spanning 1 kb of cDNA, coamplification of genomic sequences will occur rarely. Initial confirmation of the specific cDNA product depends upon careful estimation of the size of the fragment using gel electrophoresis. Spurious products can present surprisingly clean bands, but which may be the wrong size. Positive identification depends upon restriction analysis or partial sequence analysis to identify unique sequences. The presence of a known mutation in a given patient sample provides the optimum positive control and confirmation that target sequences are being amplified (Rosenzweig et al., 1991).

Contamination

Amplification of contaminating sequences, always a potential problem with PCR, is especially problematic in a protocol starting with low-copy-number target sequences and using 70 to 80 cycles (total) of amplification. To minimize this risk, positive-displacement or filtered pipet systems should be used and reagents and equipment should be segregated from general lab use. Where possible, primers should be designed such that one of each pair lies outside any plasmid subclone in use in the laboratory. PCR blanks introduced in both the reverse transcription and second PCR stages help to localize any contamination that does occur. Confirmation that contamination has been avoided is provided by finding mutations, or polymorphisms, in individual samples.

Artifacts

Taq DNA polymerase errors. Under the conditions of low template copy number, necessarily high concentrations of dNTPs, and large number of cycles, the Taq DNA polymerase error rate is increased, perhaps up to one error per 10⁴ bases (H.W., unpub. observ.). However, the vast bulk of PCR product sequences will be correct at each base position and the erroneous copies are likely to be at levels too low to interfere with screening for mutations. The one common exception to this situation occurs when products are subcloned for sequencing. In that instance, it is imperative to sequence multiple clones to distinguish true mutations from errors. Direct sequencing of PCR-amplified DNA (CPMB UNIT 15.2) obviates this problem. Theoretically, should the copy number of target mRNA molecules fall low enough (e.g., <10 to 20 per reaction), first-cycle errors will be expanded geometrically and may be detected. This appears to occur rarely, or not at all, starting with 2 µg of total RNA. Amplification of only one allele has not been observed, suggesting that copy number is never as low as a single copy (H.W., unpub. observ.). If necessary, repeating the amplification with an independent sample will eliminate any uncertainty.

Abnormal length cDNAs. Occasionally, certain RNA samples will consistently generate aberrant PCR products of incomplete length in addition to the normal product. The abnormal products will be visible by agarose gel electrophoresis. If repeat amplification from a fresh RNA isolate from the same patient sample is normal, then the aberrant band does not reflect a mutation (e.g., deletion or aberrant splicing) but presumably some artifact related to degradation of the RNA. Abnormal length products of this type may confuse analysis for mutations, by appearing as an abnormal band in RNase protection or chemical cleavage assays. If such a band correlates with an abnormal product on gel electrophoresis, subsequent amplification from a different RNA sample or gel purification of the normal size product will confirm it to be artifactual.

Genomic DNA amplification. In general, amplification from a genomic template with a single round of PCR is simpler than the two-step protocol necessary for obtaining cDNA sequences from lymphocytes. Genomic DNA samples that have been diluted, heat-denatured, and stored at 4°C are stable for years; they rarely fail as templates. The starting copy number is higher and less amplification is needed, so it is easier to control against contamination and amplification artifacts. Taq DNA polymerase errors do still occur, however, so if the PCR product is to be subcloned, analyses (e.g., sequencing) must be performed on multiple independent clones.

The positions of the oligonucleotide primers are generally dictated by the genomic structure of the target gene, particularly the exon-intron boundaries. It is these considerations that determine whether the simpler genomic DNA amplification protocol is appropriate. In a large gene with multiple exons, coding sequence is more efficiently obtained by the amplification of cDNA. Where only the cDNA sequence is known, the latter approach is the only option.

Anticipated Results

Whether from cDNA or genomic DNA template, the final PCR should yield amplification products of the predicted length, without abnormal-length products or contamination of blank samples. The PCR products are appropriate for direct analysis with the protocols described in the remaining units of this chapter.

Time Considerations

Preparation of cells, extraction of RNA (performed using a one-step protocol), and reverse transcription can be performed in 1 day. The cDNA synthesized in this way is stable overnight at 4°C or may be stored at least 1 year at 20°C. Both PCR amplifications and gel electrophoresis can be completed in 1 day. The reverse transcription and amplification stages can be performed efficiently for many samples processed in parallel (e.g., 40 samples plus controls in a typical thermal cycler).

Literature Cited

Chelly, J., Kaplan, J.-C., Maire, P., Gautron, S., and Kahn, A. 1988. Transcription of the dystrophin gene in human muscle and nonmuscle tissues. Nature 333:858-860.

Roberts, R.G., Bentley, D.R., Barby, T.F.M., Manners, E., and Bobrow, M. 1990. Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. Lancet 336:1523-1526.

Rosenzweig, A., Watkins, H., Hwang, D-S., Miri, M., McKenna, W.J., Traill, T., Seidman, J.G., and Seidman, C.E. 1991. Preclinical diagnosis of familial hypertrophic cardiomyopathy by genetic analysis of blood lymphocytes. New Engl. J. Med. 325:1753-1760.

Sarkar, B. and Sommer, S. 1989. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 244:331-334.

Watkins, H., Rosenzweig, A., Hwang, D-S., Levi, T., McKenna, W.J., Seidman, C.E., and Seidman, J.G. 1992. Distribution and prognostic significance of myosin missense mutations in familial hypertrophic cardiomyopathy. New Engl. J. Med. 326:1108-1114.

Key Reference

Sarkar, B. and Sommer, S. 1989. See above.

Describes evidence for basal rates of transcription for four tissue-specific genes in each of four tissue types.

Use of nested primers for successive rounds of PCR (see Strategic Planning). (A) Primer R may be used for reverse transcription of cDNA from an mRNA template; it is also used with primer F (forward) to generate the first-round PCR product. (B) Arrangement of nested inner primers for amplification of sections containing possible mutations.

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