EFFECT OF PCR CONDITIONS ON THE FORMATION OF HETERODUPLEX AND SINGLE-STRANDED-DNA PRODUCTS IN THE AMPLIFICATION OF BACTERIAL RIBOSOMAL DNA SPACER REGIONS
Ma. Jensen et N. Straus, EFFECT OF PCR CONDITIONS ON THE FORMATION OF HETERODUPLEX AND SINGLE-STRANDED-DNA PRODUCTS IN THE AMPLIFICATION OF BACTERIAL RIBOSOMAL DNA SPACER REGIONS, PCR methods and applications, 3(3), 1993, pp. 186-194
PCR amplifications of 16S/23S rDNA spacer regions were carried out fro
m conserved 16S and 23S sequencer for genomic DNA samples from strains
representing 16 bacterial species (12 genera). Multiple products were
produced containing conserved homologous sequences at the 3' and 5' e
nds, separated by highly variable Internal spacer sequences. These pro
ducts cross-hybridized forming heteroduplex DNA structures containing
double-stranded ends surrounding an internal single-stranded loop. Sin
gle-stranded DNA was also produced in the amplification of rDNA spacer
sequences. Fragments comprising the nonhomoduplex DNA components were
identified by their susceptibility to removal by digestion with a sin
gle-stranded endonuclease. The relative formation of heteroduplex and
single-stranded DNA was reduced by reaction conditions favoring primer
/template annealing, for example, higher ionic strength, higher primer
concentration, and lower annealing temperature, as well as by decreas
ing the number of amplification cycles. Heteroduplex and single-strand
ed DNA structures were also generated by denaturing and reannealing sp
acer amplification products in the absence of polymerase activity. Whe
reas heteroduplex and single-stranded DNA structures provide additiona
l information that is helpful in distinguishing between species of bac
teria that produce similar homoduplex products, the mobility of hetero
duplex and single-stranded DNA structures DNA structures is extremely
sensitive to electrophoretic conditions.