Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories.
Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies. The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming. We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR). Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes. The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors. As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene. We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously. It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's. Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis. Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.
Tarleton
J
J
Molecular Genetics Laboratory, Greenwood Genetic Center, South Carolina.
Schwartz
C E
CE
D001435:Bacteriophages; D004247:DNA; D006801:Humans; D007753:Laboratories; D021141:Nucleic Acid Amplification Techniques; D015341:Nucleic Acid Probes / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
D015341:Nucleic Acid Probes; D004247:DNA