Backtracking is widely used in leukemia research. In the previous blog post, I have introduced to you a little bit about backtracking. Today, I am going to describe about the backtracking method used in leukemia research.
Backtracking Target
There are many things that we need to know before we can use backtracking in leukemia research. We have to determine the choice of backtracking target according to the research question that we wish to find out the answer. For instance, there is a variety of molecular alterations that we can found in leukemic cells. These alterations may be backtracked to birth by using ANB cards.
Many translocations that can be found in childhood leukemia have helped to define the breakpoint cluster regions which that can be amended to sequencing with either long distance or long distance inverse PCR. The less well-defined rearrangements regions may need other additional techniques so that the breakpoints can be well localized. For example, the interchromosomal deletions may require Fluorescent in-situ Hybridization (FISH) or array-based Comparative Genomic Hybridization (CGH) to localize the breakpoints.
Next, the researchers need to aim for the second class of targets that defined by single base pair mutations such as RAS mutation. Although we can easily use the standard mutation detection methods to discover those targets, it is hard for the researchers to design specific assays as the inflation rate of false positives. In contrast, the IG variable regions are similar to translocations which can be backtracked. The identification and sequencing of IG variable regions are easier if compare with the single base pair mutations.
Backtracking Results
Backtracking is to find a binary answer in cancer research which is either positive result or negative result in whether the mutation is present during the birth. Most of the assays for backtracking are developed to undergo at the maximum level of sensitivity which means that it is able to detect even a single target in hundred thousand cells. Yet, the problem now is to maximize the sensitivity and minimize the false positives outcome at the same time. It is too difficult to balance these two key factors for backtracking in leukemia research. There are a lot of optimization steps that we need to pay attention.
Inadequate of Backtracking
Unfortunately, there are few questions that cannot be answered by using backtracking currently. For example, by using backtracking, we cannot get the answer for the relationship between the amount of mutations during birth and risk of leukemia or when the onset of the disease. The researchers need some refinement of techniques to give the report about that.
As backtracking assays are very sensitive, we need a good laboratory practices for polymerase chain reaction (PCR) for the best result. The special care is required when doing the secondary reactions in order to confirm there is no contamination of PCR products and patient sample on the ANB card.
Summing up, it is not easy to undergo backtracking in leukemia research. The researchers are optimizing this technique so that it can help more in leukemia research especially for childhood cancer. Maybe I will use this technique for my cancer cytogenetics research too.
(Reference: Leukemia edited by Chi Wai Eric So)
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