Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.
The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.
Afonina
I
I
Epoch Pharmaceuticals Inc., 1725 220th Street SE, #104 Bothell, WA 98021, USA. iafonina@epochpharm.com
Zivarts
M
M
Kutyavin
I
I
Lukhtanov
E
E
Gamper
H
H
Meyer
R B
RB
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011760:Pyrrolidinones / Q000378:metabolism; D013698:Templates, Genetic
C106829:1,2-dihydro-(3H)-pyrrolo(3,2-e)indole-7-carboxylate; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011760:Pyrrolidinones; D004247:DNA