TY - JOUR
T1 - A primerless molecular diagnostic
T2 - phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)
AU - Jung, Cheulhee
AU - Ellington, Andrew D.
N1 - Funding Information:
This work was supported by the National Institutes of Health [5 R01 AI092839, 5 R01 GM094933]. We sincerely thank Caitlin Sanford for her editing services.
Publisher Copyright:
© 2016, Springer-Verlag Berlin Heidelberg.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3’ terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays. [Figure not available: see fulltext.]
AB - There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3’ terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays. [Figure not available: see fulltext.]
KW - Isothermal amplification
KW - PS-THSP
KW - Phosphorothioate
KW - Self-folding
UR - http://www.scopus.com/inward/record.url?scp=84962172916&partnerID=8YFLogxK
U2 - 10.1007/s00216-016-9479-y
DO - 10.1007/s00216-016-9479-y
M3 - Article
C2 - 27032410
AN - SCOPUS:84962172916
SN - 0016-1152
VL - 408
SP - 8583
EP - 8591
JO - Fresenius Zeitschrift fur Analytische Chemie
JF - Fresenius Zeitschrift fur Analytische Chemie
IS - 30
ER -