High throughput sequencing of end regions of long linear DNAs
This invention relates to linking, amplifying and sequencing of two ends of long linear DNAs. In particular, this invention provides methods for pairing and sequencing VH and VL genes that encode two parts of one immunoglobulin. The method of the present invention can be applied to rapid antibody discovery and engineering.
1. A method for obtaining sequence identities of a plurality of clones of a phage-displayed antibody library, comprising,
providing plurality of linear double stranded DNA(dsDNA) molecules from said clones, each dsDNA molecule containing (1) a 5′ end segment at the 5′ end of the dsDNA molecule and (2) a 3′ end segment at the 3′ end of the dsDNA molecule, wherein the two end segments are separated by a spacer segment and encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, respectively;
fusing the two end segments to form a circular dsDNA molecule and a joined section on the dsDNA molecule that has the two end segments joined by a junction, and
sequencing the joined section of each dsDNA molecule from each clone using high throughput sequencing thereby obtaining the sequence identities of the plurality of clones.
2. The method of claim 1 , wherein the fusing step is carried out by a process comprising:
joining each of the 5′ and 3′ ends of the dsDNA molecule with an adaptor; and
incubating the dsDNA molecule under conditions permitting annealing of the two ends via the sequence of the adaptor.
3. The method of claim 2 , wherein the adaptor is selected from the group consisting of a palindromic adaptor, aU-containing adaptor, and a loxP adaptor.
4. The method of claim 3 , wherein the palindromic sequence is about 20 bps long.
5. The method of claim 3 , wherein the palindromic sequence has a Tm of about 50° C.
6. The method of claim 2 , wherein the adaptor is added to the two ends of the dsDNA molecule by ligation or via PCR.
7. The method of claim 1 , wherein the fusing step is carried out at 50° C. in presence of: (i) an exonuclease for generating complementary single strand overhang at each end, (ii) a polymerase to fill in a gap after overhangs at the ends hybridize to each other, and (iii) a ligase to seal a nick.
8. The method of claim 1 , before the sequencing step further comprising fragmenting the circular dsDNA molecule to generate fragments of a pre-determined size, and selecting fragments containing the two end segments.
9. The method of claim 8 , wherein the method prior to the sequencing step further includes hybridizing to the joined section with a biotin labeled probe that is complementary to the joined section and isolating the fragment that contains the joined section using streptavidin beads.
10. The method of claim 1 , before the sequencing step further comprising amplifying the joined section to generate amplicons.
11. The method of claim 10 , wherein the amplification is conducted with a forwarding primer annealing to immediately upstream of the 3′ end fragment (3′ in the original linear version), and a reverse primer anneal to immediately downstream of the 5′ end fragment (5′ in the original linear version).
12. The method of claim 11 , wherein the forward primer or the reverse primer has a tag sequence at the 5′ end.
13. The method of claim 10 , wherein the amplicons are directly sequenced.
14. The method of claim 1 , further comprising the amplifying the circular dsDNA molecule by rolling circle amplification (RCA).
15. The method of claim 14 , wherein the RCA is carried out by Phi29 in presence of dNTP and random hexamer or specific primers.
16. The method of claim 1 , wherein one or more labeled nucleotide triphosphates is incorporated into the junction in the circular dsDNA molecule.
17. The method of claim 16 , wherein one or more Biotin-dUTPs are incorporated at the junction.
18. The method of claim 17 , comprising selecting sections labeled with biotin.
19. The method of claim 1 , wherein the sequencing step is conducted by a solid phase pyrosequencing method.