Cloning a Chromosomal Rearrangement with Single Gene Sequence for Backtracking

Cloning chromosomal rearrangement with single gene sequence is totally different with the way to clone chromosomal rearrangement when both break points can be estimated. We have discussed the cloning chromosomal rearrangement when both break points can be estimated before this. Today, we are going to look through about how to clone the chromosomal rearrangement with a single gene sequence by using Inverse Polymerase Chain Reaction (IPCR) technique. The purpose we clone the chromosomal rearrangement is for leukemia cancer research by using backtracking.

Protocol to Clone Chromosomal Rearrangement by Using IPCR

Before we can proceed to IPCR, the preliminary examination of chromosomal rearrangements with FISH, karyotyping or aCGH were performed so that we can identify the breakpoint locations first. The IPCR was used to sequence the rearrangement if there was only one side of the breakpoint can be well detected. If the both sides of the breakpoint were well defined, we can just use the Long Distance Polymerase Chain Reaction (LD-PCR) which is the technique that we mentioned in the previous post.

Next, we need to design the IPCR assay. The 60Kb regions which included the 30Kb upstream and downstream surrounding the putative break in chromosome were examined. The restriction map of this region was created according to the known sequence. The restriction endonuclease that can cut at least 7Kb before the break on the retained sequence and at least 15Kb after the break on the other side of the putative breakpoint location was required.

Furthermore, two primers adjacent to the restriction site prior to the chromosomal break were required to be designed. There are some requirements for these primers.

• Length of the primers should be in 25 to 30 nucleotides.
• T_m of 68ºC.
• Spaced at 500bp intervals.

After the IPCR assay was designed, 500ng of the high molecular weight of DNA was digested with the 10 U of the chosen restriction endonuclease for 2 hours under 37ºC in the suitable buffer. The endonuclease was denatured by PCI extract the digest. The product was precipitated with 2.5 volumes of ethanol with 0.1 volume of 3M sodium acetate. 449µl of water was added to the precipitation DNA. The sample was pipette up and down gently for reconstitution. 50µl of T4 DNA ligase reaction buffer was added.

Then, the digested product was circularized by using 1µl of T4 DNA ligase in a total reaction volume of 500µl. The mixture was pipette up and down to mix well and incubated at 16ºC for 16 hours. The advantage of the diluted form of this reaction is to make sure the intramolecular ligations occur rather than intermolecular.

Later, the Qiagen kit was used after ligation for DNA extraction. The ligation reaction was transferred to a 15ml centrifuge tube. 1.5ml of buffer QG was added to the ligation reaction along with 500µl of isopropanol. The QIAquick spin column was placed in a 2ml collection tube. 500µl of the sample was added to the column and centrifuged for 1 minute at 14,000 RCF. This step was repeated for twice. Then, 30µl of buffer EB was added to the center of the membrane. The column was placed for 1 minute and centrifuged for another 1 minute at 14,000 RCF. The EDTA was added to a final concentration of 1mM to the eluted DNA in order to preserve the DNA.

Consequently, the primary LD-PCR was performed using Elongase enzyme mix. The secondary reaction was performed by using the primer set that previously used. The PCR product was sequenced to identify the exact DNA sequence of the breakpoint.

Summing up, I know this kind of scientific article is not easy to read through. However, I still wish to share out as it might benefit to people who need it especially in cancer research. Anyway, thanks for your patience to go through all of it!

(Reference: Leukemia edited by Chi Wai Eric So)