Amplification of sequences from affected individuals.
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 Basic Protocol 1 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 Basic Protocol 2 may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.
Watkins
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HC
Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
D018076:DNA, Complementary / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000235:genetics; D005826:Genetics, Medical; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D012333:RNA, Messenger