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US 5,928,906 AGranted
Process for direct sequencing during template amplification
AssigneeSequenom, Inc.
InventorsHubert Koster, Dirk Van Den Boom, Andreas Ruppert
Life of this patent
Estimating the term…
Abstract
Processes and kits for simultaneously amplifying and sequencing nucleic acid molecules, and perfonning high throughput DNA sequencing are described.
Claims48
A kit for obtaining amplified and chain terminated nucleic acid molecules from a nucleic acid template comprising;
- i) a set of chain-elongating nucleotides;
- ii) at least one chain-terminating nucleotide;
- iii) a first polymerase;
- and iv) a second polymerase, which has a higher affinity towards at least one chain terminating nucleotide relative to the first polymerase.
1 more element
A process for simultaneously amplifying and sequencing a single stranded nucleic acid molecule, comprising the steps of:
- a) contacting the single stranded nucleic acid molecule with:
- i) at least one primer that can hybridize to the single stranded nucleic acid molecule, (ii) a set of chain elongating nucleotides, (iii) at least one chain terminating nucleotide, (iv) a first DNA polymerase;
- and (v) a second DNA polymerase, which has a higher affinity towards the chain terminating nucleotide relative to the first polymerase, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
- b) detecting the chain terminated fragments by a detection means;
- and
- c) aligning the fragments to determine the sequence of the single stranded nucleic acid molecule.
3 more elements
A process for simultaneously amplifying and sequencing a nucleic acid molecule, comprising the steps of:
- a) denaturing a double stranded DNA molecule to obtain two complementary single stranded DNA molecules;
- b) contacting at least one of the single stranded DNA molecules with a complementary primer to obtain at least one primer containing single stranded DNA molecule;
- c) contacting the at least one primer containing single stranded DNA molecule with:
- (i) a set of chain elongating nucleotides, (ii) at least one chain terminating nucleotide, (iii) a first DNA polymerase;
- and (iv) a second DNA polymerase, which has a higher affinity towards the chain terminating nucleotide relative to the first polymerase, so that polymerization by the first polymerase results in amplification and polymerization by the second polymerase results in the formation of chain terminated fragments;
- d) repeating steps a) through c) for an appropriate number of times to obtain chain terminated fragments for detection;
- and
- e) detecting the chain terminated fragments by a detection means and aligning the fragments to determine the sequence of the nucleic acid molecule.
5 more elements
Dependencies are inferred from the claim text.