Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.
1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.
Hormonal Bioregulation Laboratory, Laval University Hospital Research Centre, Ste-Foy, Qudébec, Canada.
P
P
Chapdelaine
M A
MA
Ho-Kim
R R
RR
Tremblay
J Y
JY
Dube
D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods*; D011232:Chemical Precipitation; D004247:DNA / Q000302:isolation & purification; D000431:Ethanol; D006801:Humans; D007610:Kallikreins / Q000032:analysis* / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D011467:Prostate / Q000032:analysis*; D017430:Prostate-Specific Antigen; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D000951:Antigens, Neoplasm; D009841:Oligonucleotides; D012333:RNA, Messenger; D000431:Ethanol; D004247:DNA; D012343:RNA, Transfer; D007610:Kallikreins; D017430:Prostate-Specific Antigen