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Nucleic Acids Research, 1993, Vol. 21, No. 21 4983 -4984
A rapid method for sequencing trinucleotide repeats
Russell L.Margolis1, Shi-Hua Li1'2 and Christopher A.Ross1 2 Laboratory of Molecular Neurobiology, Departments of 'Psychiatry and 2Neuroscience, Johns Hopkins University School of Medicine, 618 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205, USA Received August 20, 1993; Accepted September 16, 1993
A newly discovered form of human mutation, trinucleotide repeat expansion, is now known to cause five diseases and may cause other diseases with unusual forms of inheritance (1). Certain neoplasms may also stem from trinucleotide repeat expansion in somatic cells (2, 3) and trinucleotide repeats are present in a number of genes coding for transcription factors (1). Genes containing trinucleotide repeats are therefore of intrinsic biological interest and may serve as candidates for the etiology of a variety of diseases. We and others have attempted to identify novel genes containing trinucleotide repeats by screening cDNA libraries with inserts of sufficient length to reveal reading frame (4, 5). Characterization of the trinucleotide repeat in these DNA fragments necessitates sequencing until the repeat is reached, a time consuming and expensive process. By using oligonucleotide primers composed of the repeat with a degenerate 3' non-motif base, flanking regions of dinucleotide (CA) repeats have been successfully sequenced, and the complement of this sequence used to design primers for sequencing back through the repeat (6). To expedite the process of characterizing trinucleotide repeats, we have developed a similar method for direct sequencing of regions flanking trinucleotide repeats. The method utilizes unique sets of degenerate primers and unique primer-template annealing temperatures (since repeat G-C content varies from 0% to 100%) for each type of trinucleotide repeat. Clones containing trinucleotide repeats were identified by screening a human cerebral cortex cDNA library in lambda ZAP (Stratagene) with 30mer or 60mer oligonucleotides containing 10 or 20 CTG, AAT, or CCG repeats, using standard techniques as we have previously described (4). Positives were plaque purified, and plasmids (pBluescript SK) were isolated using an in vivo excision procedure (Stratagene). Separate screenings were
performed with each oligonucleotide probe. Though for convenience repeats have been termed CTG, AAT, and CCG, each actually represents 6 possible reading frames (e.g., an AAT probe will identify repeats with reading frames AAT, ATA, TAA, TTA, TAT, and ATT). Plasmids (5 yg per reaction) were denatured by adding 0.1 volume of 2 M NaOH and 2 mM EDTA and incubating for 30 minutes at 37°C. After neutralization with 0.1 volume of 3 M sodium acetate (pH 4.5 -5.5), the DNA was precipitated by adding 3 volumes of 100% ethanol and chilling at -70°C for 15 minutes. DNA was pelleted by microcentrifugation, rinsed with 70% ethanol, repelleted, dried and suspended in 7 Al H20. The first step in the standard dideoxynucleotide sequencing protocol (Sequenase Version 2.0 DNA Sequencing Kit, U.S. Biochemical) is to anneal primer to
denatured template (1:1 molar ratio) in the presence of reaction buffer (40 mM Tris-HCI, 20 mM MgCl2, 50 mM NaCl final concentration) by heating, slowly cooling, and then placing on ice. We modified this procedure in two ways to obtain sequence flanking trinucleotide repeats. First, l9mer or 21mer oligonucleotide primers were designed which would specifically hybridize to the 3' end of the repeats of interest by including
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