A rapid method for PCR amplification of DNA directly from cells fixed in Carnoy's fixative.
We describe a method for rapid and efficient polymerase chain reaction (PCR) amplification of specific target DNA sequences directly from cells fixed in 3:1 methanol-acetic acid (Carnoy's fixative) for routine cytogenetic analysis. The fixed cells used had been stored at -20 degrees C from a few weeks up to 6 years. Primer sets used correspond to loci on an autosome (retinoblastoma, RB1), as well as the X (Duchenne muscular dystrophy, DMD) and Y (sex-determining region of the Y, SRY) chromosomes. Sizes of amplified products were the expected 400, 251 and 609 bps, respectively. No differences in quality of amplification products were found between PCR templates obtained from fresh tissues or from cells fixed for varying lengths of time in Carnoy's fixative. This technique has the following advantages: (1) it allows retrospective studies of genetic disorders from archived specimens; (2) it requires only a limited number of cells; (3) it is rapid and simple; and (4) it avoids multistep procedures required in extraction of the DNA.
Li
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Department of Medical Genetics, University of South Alabama, College of Medicine, Mobile 36688-0002, USA.
Tuck-Muller
C M
CM
Yan
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Wertelecki
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W
Chen
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H
D000085:Acetates; D019342:Acetic Acid; D001483:Base Sequence; D002725:Chloroform; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D004247:DNA / Q000097:blood*; D017931:DNA Primers; D000431:Ethanol; D005260:Female; D005404:Fixatives; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012730:Sex Chromosomes / Q000235:genetics
D000085:Acetates; C033474:Carnoy's solution; D017931:DNA Primers; D005404:Fixatives; D000431:Ethanol; D002725:Chloroform; D004247:DNA; D019342:Acetic Acid