Amplification of Sequences from Affected Individuals

Hugh C. Watkins1

1 Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.1
DOI:  10.1002/0471142905.hg0701s00
Online Posting Date:  May, 2001
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Abstract

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). The first describes harvesting of 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 may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Amplification of RNA from Lymphocytes
  • Alternate Protocol 1: Modified Second‐Round Amplification of cDNA
  • Basic Protocol 2: Amplification of Genomic DNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Amplification of RNA from Lymphocytes

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; 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 2D), ice cold
  • recipeDiethylpyrocarbonate (DEPC)‐treated H 2O (see recipe)
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 25‐mer oligonucleotide primers (Fig. ): 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 for guidelines on standard methods to protect against contaminating RNases.

Alternate Protocol 1: Modified Second‐Round Amplification of cDNA

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Genomic DNA ( appendix 3B)
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 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)
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Figures

Videos

Literature Cited

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.
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