The use of polymerase chain reaction generated nucleotide sequences as probes for hybridization.
In this report a rapid, simple and economical means of preparing a cDNA probe of specified length, sequence and specific activity is described. The process involves the use of a polymerase chain reaction to incorporate radiolabelled nucleotides into a single stranded or double stranded cDNA sequence. A pair of oligonucleotide primers are synthesized, flanking the sense and antisense strands of a selected sequence. The primers are then used with a cloned DNA fragment or a cellular source of RNA or DNA as a template to amplify the specific gene sequence. The sequence to be used as a probe is selected from the known sequence using free energy calculations of the secondary structure. The calculation of free energy predicts regions of stable secondary structure which may hinder transcription and thus are to be avoided. By selecting the distance between primers the probe length can be controlled to allow adequate probe permeability into tissue samples. The specific region of the gene sequence can be chosen to differentiate between closely related sequences by avoiding areas of homology. Altering the concentration of a radiolabelled nucleotide allows direct control of probe specific activity. The use of asymmetric PCR allows the preferential generation of an antisense single stranded cDNA sequence for a higher sensitivity in the detection of low abundance mRNA. This report highlights the advantage of this technique in generating probes for in situ hybridization. However, any technique that relies on homology for detection of sequences, such as Northern and Southern blotting could also utilize this technique.
Scully
S P
SP
Orthopedic Research Unit, NIAMS NIH, Bethesda, MD 20892.
Joyce
M E
ME
Abidi
N
N
Bolander
M E
ME
D000818:Animals; D001483:Base Sequence; D003094:Collagen / Q000235:genetics*; D015342:DNA Probes; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D051381:Rats
D015342:DNA Probes; D012333:RNA, Messenger; D003094:Collagen