Transposon mediated multiplex sequencing
View Patent ↗The present invention relates to an automated method of transposon-mediated multiplex sequencing of DNA fragments inserted into a vector. It relates more particularity to an increased efficiency in such automated methods, where the increased efficiency is obtained by screening out before the sequencing those constructs in which the transposon inserted into the vector sequence. This prevents a waste of time and resources in performing reactions sequencing the vector instead of the DNA fragments of interest.
1. A process for parallel, transposon-mediated sequencing of DNA, the process comprising:
i) providing one or more target DNA sequences, each inserted into a vector at an insertion site;
ii) amplifying each target DNA-containing vector and pooling them, wherein each target DNA sequence is represented in the pool in an substantially equal amount per kb;
iii) exposing the pool of target DNA-containing vectors to a selectable transposon, wherein the selectable transposon integrates into the target DNA-containing vectors at substantially random sites to form a pool of target DNA- and transposon-containing vectors;
iv) transforming cells with the pool of target DNA- and transposon-containing vectors and isolating and growing a representative number of individual transformants into cultures under selection conditions;
v) performing a polymerase chain reaction on DNA from each culture, wherein the polymerase chain reaction uses primers complementary to the 3′ ends of the vector sequence at the insertion site and has an extension time during each reaction cycle of the polymerase chain reaction sufficient to efficiently produce a DNA with the size of a full-length of the vector sequence but too short to efficiently produce a DNA with the size of a full-length of the vector sequence plus the transposon sequence;
vi) measuring the amount of DNA produced in each polymerase chain reaction;
vii) selecting cultures whose polymerase chain reactions produced substantial amounts of DNA above a cutoff value; and
viii) sequencing the transposon flanking regions of the target DNA-containing vector from the selected cultures.
2. The process of claim 1 , wherein one or more of steps i) to viii) are automated.
3. The process of claim 1 , wherein the step of measuring the amount of DNA produced in each polymerase chain reaction comprises adding a fluorescent stain selective for dsDNA to the finished polymerase chain reaction and measuring the resulting amount of fluorescence.
4. The process of claim 3 , wherein one or more of steps i) to viii) are automated.
5. The process of claim 3 , wherein the fluorescent stain selective for dsDNA is selected from the group consisting of PICOGREEN® and bisbenzimide dyes.
6. The process of claim 3 , wherein the fluorescent stain selective for dsDNA is PICOGREEN®.
7. The process of claim 1 , wherein the vector and transposon carry different selectable markers.
8. The process of claim 1 , wherein the transposon is at least 3 kb.
9. The process of claim 8 , wherein the transposon is at least 4 kb.
10. The process of claim 9 , wherein the transposon is GPS-Apra-2 um-URA3.
11. The process of claim 8 , wherein the transposon is at least 5 kb.
12. The process of claim 1 , wherein the transposon is equal to or greater than the vector in length.
13. The process of claim 12 , wherein the transposon is at least about 5/4 the length of the vector.
14. The process of claim 13 , wherein the transposon is about 5 kb and the vector is about 4 kb.
15. The process of claim 12 , wherein the transposon is at least about 5/3 the length of the vector.
16. The process of claim 15 , wherein the transposon is about 5 kb and the vector is about 3 kb.