Chromosomal rearrangement is a common genetic abnormality. Some of the examples of chromosomal rearrangements like translocations and deletions occur in a single breakpoint but two strands of DNA. This kind of chromosomal rearrangement requires the broken form of double-strands DNA.
Today, we are going to explore about the cloning chromosomal rearrangement for backtracking in leukemia cancer research. Therefore, we need to know the tools or techniques that can be used to locate the breakpoint of the chromosomal rearrangement.
Translocations in Chromosomal Rearrangement
Firstly, we will discuss about the method used to locate the breakpoint of chromosomal translocations. If the partner of translocation has been detected via Fluorescence in situ Hybridization (FISH) or karyotype, we need to use a long distance polymerase chain reaction (LD-PCR) approach to locate the breakpoint. As I mentioned before, FISH is a tool for the detection of significant chromosomal abnormalities in childhood leukemia. We need this technique for leukemia cancer research.
Deletions in Chromosomal Rearrangement
On the other hand, if a deletion without apparent partner was identified by using FISH or array-comparative genomic hybridization (aCGH), we can use the inverse PCR (IPCR), a one-sided method to identify the breakpoint. As I mentioned before, aCGH is a tool to analyze the leukemia genome. This tool is important for cancer research.
Protocol to Detect the Breakpoint in Chromosomal Rearrangement
1. First of all, the location of breakpoints on either side was estimated by using the known exon and intron boundaries. Besides, the techniques like high resolution aCGH can be used to estimate the location of breakpoints too.
2. The PCR primers were designed at 5 to 10Kb intervals to the left and right hand sides respectively of the predicted breakpoint. There are many requirements for the primers.
- The length of the primers should be 25 to 35bp.
- The primers have Tm of 68ºC.
- The 5’ to 3’ direction of the primers have to be directed towards the putative breakpoint.
- The primers were composed of unique sequence in human genome without runs of 4 or more mononucleotides.
Another thing that we need to take precaution is the Tm should be salt adjusted to a typical PCR buffer of 50mM Na+. Multiple primers might be required and spaced at 5 to 10Kb intervals if the estimated regions of breakpoint were larger than 10Kb or not well-defined.
3. Next, the LD-PCR was performed as recommended by the manufacturer of the polymerase. The master mix has to be set up. 100ng of DNA template was added to the reaction. If the DNA is inadequate or there are multiple primer pairs were required due to not well defined breakpoints, the primer sets may be combined, as many as three forward and three reverse in one reaction. The standard LD-PCR was run. By using electrophoresis, the 5 to 10µl of sample was visualized.
4. The PCR products were evaluated as the LD-PCR might produce false bands from mishybridization. The PCR bands will be cut out of the gel and purified by using Qiagen gel purification kit. The PCR bands were sequenced according tho the same primers used for the PCR. It there were authentic sequences produced by both primers consistent with the predetermined breakpoint location, the band can be considered as a “bona-fide” PCR band.
5. Last but not least, a set of PCR primers internal were designed to one primer at 1Kb intervals toward the putative breakpoint. The new primers were tested with the single reverse primer by using PCR products to determine the exact location of the breakpoint. The smallest product produced was sequenced and we can successfully clone the chromosomal rearrangement region for backtracking.
Summing up, cloning the chromosomal rearrangement region for backtracking requires a lot of PCR techniques and approaches. We have to familiar with the PCR so that we can do it properly.
(Reference: Leukemia edited by Chi Wai Eric So)
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