Methods of amplifying nucleic acid sequences mediated by transposase/transposon DNA complexes
Methods are provided for nucleic acid amplification including contacting a double stranded nucleic acid with transposases bound to transposon DNA, wherein the transposon DNA includes a transposase binding site and an RNA polymerase promoter sequence, wherein the transposases/transposon DNA complex bind to target locations along the double stranded nucleic acid and cleave the double stranded nucleic acid into a plurality of double stranded fragments, with each double stranded fragment having the transposon DNA bound to each 5′ end of the double stranded fragment, extending the double stranded fragments along the transposon DNA to make double stranded extension products having double stranded RNA polymerase promoter sequences at each end, contacting the double stranded extension products with an RNA polymerase to make a plurality of RNA transcripts of each double stranded extension product, reverse transcribing the RNA transcripts into single stranded copy DNA, forming complementary strands to the single stranded copy DNA to form a plurality of double stranded DNA amplicons corresponding to each double stranded fragment.
1. A method of single cell whole genome amplification and sequencing comprising
contacting double stranded genomic DNA from a single cell with Tn5 transposases each complexed with a transposon DNA, wherein the transposon DNA includes a double-stranded 19 bp Tnp binding site and an overhang, wherein the overhang includes a barcode region, a priming site and a strong T7 promoter sequence, wherein the Tn5 transposase/transposon DNA complex bind to target locations along the double stranded genomic DNA cleaving the double stranded genomic DNA into a plurality of double stranded fragments, with each double stranded fragment having a first complex attached to an upper strand by the Tnp binding site and a second complex attached to a lower strand by the Tnp binding site,
removing the Tn5 transposases from the complex,
extending the double stranded fragments along the transposon DNA to make a double stranded extension product having T7 promoters at each end,
contacting the double stranded extension product with T7 RNA polymerase to make RNA transcripts of the double stranded extension product,
reverse transcribing the RNA transcript into single stranded DNA, and
forming a complementary strand to the single stranded DNA to form a double stranded DNA including the genomic DNA sequence and having barcodes at both ends of the upper and lower strands.
2. A method of nucleic acid amplification comprising
contacting a double stranded nucleic acid with transposases bound to transposon DNA, wherein the transposon DNA includes a transposase binding site, wherein the transposase/transposon DNA complex bind to target locations along the double stranded nucleic acid and cleave the double stranded nucleic acid into a plurality of double stranded fragments, with each double stranded fragment having the transposon DNA bound to each 5′ end of the double stranded fragment,
extending the double stranded fragments along the transposon DNA to make double stranded extension products at each end, and
amplifying the double stranded extension products.
3. The method of claim 2 wherein the double stranded nucleic acid is an isolated double stranded nucleic acid and the transposase is an isolated transposase.
4. The method of claim 2 wherein the double stranded nucleic acid is genomic DNA.
5. The method of claim 2 wherein the double stranded nucleic acid is genomic DNA obtained from a single cell.
6. The method of claim 2 wherein the double stranded nucleic acid is the whole genome of a single cell.
7. The method of claim 2 wherein the transposase is Tn5 transposase.
8. The method of claim 2 wherein the transposon DNA further includes a barcode sequence and/or a priming site.
9. The method of claim 2 wherein the transposon DNA includes a double-stranded 19 bp Tnp binding site and an overhang, wherein the overhang includes a barcode region and a priming site.
10. The method of claim 2 wherein the transposon DNA includes a double-stranded 19 bp Tnp binding site and a nucleic acid loop structure including a barcode region and a priming site.
11. The method of claim 2 wherein bound transposases are removed from the double stranded fragments before extending the double stranded fragments.
12. The method of claim 2 wherein the transposases are Tn5 transposases each complexed with a transposon DNA, wherein the transposon DNA includes a double-stranded 19 bp Tnp binding site and an overhang, wherein the overhang includes a barcode region and a priming site, wherein the Tn5 transposase/transposon DNA complex bind to target locations along the double stranded genomic DNA cleaving the double stranded genomic DNA into a plurality of double stranded fragments.
13. The method of claim 2 further including the step of sequencing the double stranded extension products.
14. The method of claim 2 further including the step of detecting single nucleotide variations, detecting copy number variations, or detecting structural variations in the double stranded extension products.
15. The method of claim 2 wherein the double stranded nucleic acid is genomic DNA from a prenatal cell, a cancer cell, or a circulating tumor cell.
16. The method of claim 2 wherein the double stranded nucleic acid is genomic DNA from a single prenatal cell, a single cancer cell, or a single circulating tumor cell.
17. The method of claim 2 wherein the transposon DNA further comprises an RNA polymerase promoter sequence and wherein the double stranded extension products comprise double stranded RNA polymerase promoter sequences at each end.
18. The method of claim 17 wherein the RNA polymerase promoter sequence is a T7 promoter sequence.
19. The method of claim 17 further comprising
contacting the double stranded extension products with an RNA polymerase to make a plurality of RNA transcripts of each double stranded extension product,
reverse transcribing the RNA transcripts into single stranded copy DNA, and
forming complementary strands to the single stranded copy DNA to form a plurality of double stranded DNA amplicons corresponding to each double stranded fragment, wherein the double stranded DNA amplicons are linearly amplified from the original double stranded fragments.
20. The method of claim 19 wherein the RNA polymerase is T7 RNA polymerase.