EP 0595982 A4 19951011 - SINGLE STEP AMPLIFICATION AND SEQUENCING OF NUCLEIC ACIDS
Title (en)
SINGLE STEP AMPLIFICATION AND SEQUENCING OF NUCLEIC ACIDS
Publication
Application
Priority
- AU 9200372 W 19920723
- AU PK740091 A 19910724
Abstract (en)
[origin: WO9302212A1] Method for the amplification and sequencing of DNA or RNA. The method comprises the steps of (i) melting a double stranded nucleic acid to yield a pair of complementary nucleic acid strands, (ii) hybridising a primer to each of the strands, the primers being so chosen that the primer annealing to the sense strand is 3' to the position of the primer on the antisense strand, one of the primers being labelled so as to be capable of being visualized independently of the other primer, (iii) causing a polymerase enzyme to amplify the nucleic acid in the presence of a dideoxynucleotide analogue of one of the nucleotides present in the nucleic acid, the dideoxy analogue being present in such a concentration that a majority of the newly synthesised nucleic acid strands are terminated by incorporation of dideoxynucleotides without extending far enough to act as templates for synthesis of the opposite strand using the second primer, (iv) repeating steps (i) to (iii) sequentially a number of times, (v) repeating the steps (i) to (iv) using at least another two dideoxynucleotide analogues of the other three nucleotides present in the nucleic acid, and (vi) electrophoretically separating the reaction products of each of the repetitions of steps (i) to (iv) and visualizing the labelled strands. The other of the nucleotides of at least a part of the strand of the nucleic acid to which the labelled primer annealed between the binding sites may be determined by comparing the separated and visualized gels for each of the nucleotide analogues used.
IPC 1-7
IPC 8 full level
C12Q 1/68 (2006.01); C12Q 1/6869 (2018.01)
CPC (source: EP)
C12Q 1/6869 (2013.01)
Citation (search report)
- [A] EP 0409078 A2 19910123 - BASF AG [DE]
- [X] G. RUANO AND K.K. KIDD: "Coupled amplification and sequencing of genomic DNA", PROC. NATL.ACAD SCI., vol. 88, no. 7, 1 April 1991 (1991-04-01), NATL. ACAD SCI.,WASHINGTON,DC,US;, pages 2815 - 2819
- [A] D.B. OLSEN AND F. ECKSTEIN: "Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing", NUCLEIC ACIDS RESEARCH, vol. 17, no. 21, 11 November 1989 (1989-11-11), IRL PRESS LIMITED,OXFORD,ENGLAND, pages 9613 - 9620
- [A] K.L. NAKAMAYE ET AL.: "Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-triphosphates", NUCLEIC ACIDS RESEARCH, vol. 16, no. 21, IRL PRESS LIMITED,OXFORD,ENGLAND, pages 9947 - 9959
- [T] S.-J. DENG ET AL.: "Simultaneous amplification and sequencing of genomic DNA (SAS): sequencing of 16S rRNA genes using total genomic DNA from Butyrivibrio fibrisolvens, and detection and genotyping of nonculturable mycoplasma-like organisms directly from total DNA isolated from infected plants", J. MICROBIOL. METHODS, vol. 17, AMSTERDAM, NL, pages 103 - 113
- See references of WO 9302212A1
Designated contracting state (EPC)
AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE
DOCDB simple family (publication)
WO 9302212 A1 19930204; CA 2114124 A1 19930204; EP 0595982 A1 19940511; EP 0595982 A4 19951011; JP H07500004 A 19950105
DOCDB simple family (application)
AU 9200372 W 19920723; CA 2114124 A 19920723; EP 92916411 A 19920723; JP 50249093 A 19920723