Article

A universal nucleoside for use at ambiguous sites in DNA primers.

Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Nature (impact factor: 36.28). 07/1994; 369(6480):492-3. DOI:10.1038/369492a0 pp.492-3
Source: PubMed

ABSTRACT A non-discriminatory base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3') is as effective as the exact match d(5'-CGT AAT CAG AAA ACA AT-3'). It is also possible to sequence using a primer containing M at several contiguous positions, for example d(5'-CGT AAT MMM MMM MMM AT-3'). Melting curves show that duplexes formed on hybridization of the sequences d(5'-CCT TTT TMT TTT TGG-3') and d(5'-CCA AAA AXA AAA AGG-3'), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (Tm range 3 degrees C).

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Keywords

contiguous positions
 
corresponding duplexes
 
d(5'-CCA AAA AXA AAA AGG-3')
 
degeneracy
 
different X bases
 
exact match d(5'-CGT AAT CAG AAA ACA AT-3')
 
fragmentary peptide sequence data
 
hydrogen-bonding interactions
 
lower temperature
 
maximizes stacking
 
Melting curves
 
non-discriminatory base analogue
 
oligonucleotide probes
 
Oligonucleotides
 
polymerase chain reaction
 
previous universal nucleoside candidates
 
sequences d(5'-CCT TTT TMT TTT TGG-3')
 
sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3')
 
sterically
 
Tm range 3 degrees C
 

R Nichols