The invention relates to a method for synthesising long nucleic acids, including at least one cycle of elongating initial fragments of nucleic acids, including a) a phase comprising the enzymatic addition of nucleotides to said fragments, b) a phase comprising the purification of the fragments having a correct sequence, c) an optional phase of enzymatic amplification, each cycle being performed in a reaction medium which is compatible with enzymatic addition and amplification, such as an aqueous medium, the synthesis method also comprising, at the end of all the elongation cycles, a last step of final amplification. The invention also relates to the use of the method for the production of genes, or sequences of synthetic nucleic acids, DNA or RNA. The invention further relates to a kit for implementing said method.

The invention relates to a method for synthesising long nucleic acids, including at least one cycle of elongating initial fragments of nucleic acids, including a) a phase comprising the enzymatic addition of nucleotides to said fragments, b) a phase comprising the purification of the fragments having a correct sequence, c) an optional phase of enzymatic amplification, each cycle being performed in a reaction medium which is compatible with enzymatic addition and amplification, such as an aqueous medium, the synthesis method also comprising, at the end of all the elongation cycles, a last step of final amplification. The invention also relates to the use of the method for the production of genes, or sequences of synthetic nucleic acids, DNA or RNA. The invention further relates to a kit for implementing said method.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.

Methods and materials for preparing DNA complementary to poly(A)-minus RNA are provided. The method includes conducting a template switching reaction by contacting a RNA-cDNA intermediate with a template switching oligonucleotide (TSO), extending the cDNA strand to include sequence complementary to the TSO and degrading the TSO. The method is capable of assaying a broad spectrum of coding and non-coding RNA from a single cell.

Methods and materials for preparing DNA complementary to poly(A)-minus RNA are provided. The method includes conducting a template switching reaction by contacting a RNA-cDNA intermediate with a template switching oligonucleotide (TSO), extending the cDNA strand to include sequence complementary to the TSO and degrading the TSO. The method is capable of assaying a broad spectrum of coding and non-coding RNA from a single cell.

Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

An effective sequencing method, the method comprising: (1) performing first sequencing on a sequencing template on a chip surface, so as to facilitate obtaining first sequencing data by means of first newly generated sequencing strands being formed, the sequencing template being connected onto the chip surface by means of a sequencing adapter; (2) performing first blocking treatment on 3′ ends of at least a portion of the first newly generated sequencing strands; and (3) performing second sequencing on the sequencing template, so as to facilitate obtaining second sequencing data by means of second newly generated sequencing strands being formed.

An effective sequencing method, the method comprising: (1) performing first sequencing on a sequencing template on a chip surface, so as to facilitate obtaining first sequencing data by means of first newly generated sequencing strands being formed, the sequencing template being connected onto the chip surface by means of a sequencing adapter; (2) performing first blocking treatment on 3′ ends of at least a portion of the first newly generated sequencing strands; and (3) performing second sequencing on the sequencing template, so as to facilitate obtaining second sequencing data by means of second newly generated sequencing strands being formed.