Method of nucleic acid sequencing
OwnerEric H. Kawashima, Laurent Farinelli +1inventors
InventorsERIC H. KAWASHIMA, LAURENT FARINELLI, PASCAL MAYER
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Abstract
Different nucleic acid molecules present at different locations can be sequenced in parallel Primers that are annealed to the nucleic acid molecules can be provided. Each location can then be provided with a nucleic acid polymerase and a nucleotide. It can then be determined whether or not the nucleotide has been used in primer extension and the process can be repeated. As an alternative to using primers, a nick in a double stranded nucleic acid molecule can provide a 3′-OH group for chain extension.
Claims24
A method for sequencing nucleic acid molecules, comprising the steps of:
- a) providing at a first location a plurality of single stranded nucleic acid molecules that have the same sequences as one another and that are hybridised to primers in a manner to allow primer extension in the presence of nucleotides and a nucleic acid polymerase;
- b) providing at a second location, which is different from the first location, a plurality of single stranded nucleic acid molecules that have the same sequences as one another, but that have different sequences from the sequences of the single stranded nucleic acid molecules at the first location, and that are also hybridised to primers in a manner to allow primer extension in the presence of nucleotides and a nucleic acid polymerase;
- c) providing each location with a nucleic acid polymerase and a given labelled nucleotide under conditions that allow extension of the primers if a complementary base or if a plurality of such bases is present at the appropriate position in the single stranded nucleic acid molecules;
- d) detecting whether or not said labelled nucleotide has been used for primer extension at each location by determining whether or not the label present on said nucleotide has been incorporated into extended primers;
- e) repeating steps c) and d) one or more times so that extended primers comprising a plurality of labels are provided.
A method according to any preceding claim, wherein if the given nucleotide has been used in primer extension in step d) then this step includes the step of detecting how many of the given nucleotides have been used per extended primer.
A method according to any preceding claim, wherein after step c) excess nucleotides that have not been used in primer extension are removed (e.g. by washing).
A method according to any preceding claim, wherein step d) uses absorption or emission spectrometry.
A method according to any preceding claim, wherein said single stranded nucleic acid molecules, said primers or both of the aforesaid are immobilised.
A method according to any preceding claim that is used to fully or partially sequence 10 or more nucleic acid molecules having different sequences simultaneously.
A method according to any preceding claim that is used to fully or partially sequence 100 or more nucleic acid molecules having different sequences simultaneously.
A method according to any preceding claim that is used to fully or partially sequence 1000 or more nucleic acid molecules having different sequences simultaneously.
A method according to any preceding claim, wherein each of four different nucleotides is used in primer extension.
A method according to any preceding claim, wherein the detection step is carried out without moving the nucleic acid molecules from the different locations.
A method as described in any preceding claim with the exception that double stranded nucleic acid molecules having nicks therein are provided at the first and/or second locations instead of providing single stranded molecules hybridised to primers.
A method as described in any preceding claim with the exception that only one nucleic acid molecule is provided at the first and/or second locations.
A method for sequencing nucleic acid molecules, comprising the steps of:
- a) providing at a first location a plurality of single stranded nucleic acid molecules that have the same sequences as one another and that are hybridised to primers in a manner to allow primer extension in the presence of nucleotides and a nucleic acid polymerase;
- b) providing at a second location, which is different from the first location, a plurality of single stranded nucleic acid molecules that have the same sequences as one another, but that have different sequences from the sequences of the single stranded nucleic acid molecules at the first location, and that are also hybridised to primers in a manner to allow primer extension in the presence of nucleotides and a nucleic acid polymerase;
- c) providing each location with a nucleic acid polymerase and a given nucleotide in labelled and unlabelled form under conditions that allow extension of the primers if a complementary base or if a plurality of such bases is present at the appropriate position in the single stranded nucleic acid molecules;
- d) detecting whether or not said labelled nucleotide has been used for primer extension at each location by determining whether or not the label present on said nucleotide has been incorporated into extended primers;
- e) repeating steps c) and d) one or more times.
A method of sequencing a target nucleic acid comprising:
- (a) hybridizing the target nucleic acid to a primer whereby the target nucleic acid can serve as a template for extension of the 3′end of the primer;
- (b) incubating the hybridized target nucleic acid/primer with a polymerase and a type of nucleotide bearing a label under conditions supporting template-directed extension of the primer if the nucleotide type can be incorporated as the complement of a corresponding nucleotide of the target;
- (c) measuring first label incorporated into the primer to determine whether, and if so, by how many base increments, the primer has been extended by incorporated of the nucleotide type;
- (d) incubating the hybridized primer/target nucleic acid with a different type of nucleotide bearing a label under conditions supporting template-directed extension of the primer if the different nucleotide type can be incorporated so as to be complementary to a corresponding nucleotide in the target;
- (e) measuring incremental label incorporated into the primer due to the previous incubating step to determine whether, and if so, by how many base increments, the primer has been extended by incorporation of the different nucleotide type;
- and
- (f) repeating steps (b)-(e) until a desired portion of the target sequence can be determined from the incremental base additions to the primer.
The invention substantially as hereinbefore described.
Dependencies are inferred from the claim text.