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Annotated Bibliography for Master Student

Chattopadhyay A,  Redner R. L. Cryptic insertion of PML-RARA into the 3p25 locus in an acute promyelocytic leukemia with t(3;17)(p25;q21). Cancer Genetics and Cytogenetics 2010; 201 (2010): 28-31.

In the 2010 Cryptic Insertion of PML-RARA Into The 3p25 Locus In An Acute Promyelocytic Leukemia with t(3;17)(p25;q21) journal article, authors Robert L. Redner and Anuja Chattopadhyay describe the rationale, methodology, as well as the findings of a Acute promyelocytic leukemia (APL) bone marrow study the 2 carried out.

1 Further, the scientists form conclusions that indicate the need for new karyoptic abnormalities to be analyzed at the molecular level. To express their motivation for the study, the researchers describe several cases wherein patients demonstrated RARA (retinoic acid receptor alpha) translocation variations. The authors own up that such rare variations account for less than twenty-percent of APL, they state that such figures are adequate bases for testing APL molecular origins.

Regarding the methodology, enrichment, pelleting, and -80oC freezing was successively done on bone marrow mononuclear cell fraction.

A Blood and Cell Culture DNA Kit was used to derive genomic DNA from the marrow followed by whole-genomic amplification via REPLI-g kit. RARA intron 2 configuration was studied via Southern blot experiment. Restriction enzyme KpnI was used to digest genomic DNA (20 µg) before electrophoresis fractionation on some one-percent agarose gel. Afterwards, depurination, denaturation and  neutralization was one on the DNA before being moved to a nylon film. Successive hybridization was done on the blot using three 32P- tagged RARA probes, matching with sequences 13730-14278, 1186-12330, and 5684-6405 16,919-bp-long RARA intron 2 probe 3.

 In deriving the panhandle, KpnI was used to digest genomic DNA before cleaving dephosphorylation using calf intestinal alkaline phosphatase. Consequently, A one-stranded 5’ phosphorylated adaptor oligonucleotide was ligated onto the DNA. The DNA polymerase I’s  Klenow fragment was used to fill the  adaptor’s complimentary strand.  Fifty µL of  reaction mixture having two-hundred-and-fifty adaptor-ligated DNA, five 10×PCR buffer µL, and two-hundred µmol/L NTPS was warmed to ninety-four 0C. The mixture wads then cooled to 72 0C to allow the ligated oligo-to-RARA sequence complementary strand to anneal towards the 5’ extremity. The reaction was continued at seventy-two 0C for thirty seconds. Afterwards, two-hundred nmol/L RARA PH-2 and RARA PH-1 panhandle primers were introduced into the reaction. Successive denaturation was employed to amplify the division region. Primer set RARA PH-3 and I-µL primary PCR aliquot were used to conduct  nested PCR. Sub-cloning of PCR products into a Carlsbad, Invitrogen, and CA Topo vector was done. The resulting sequence was analyzed.

Regarding the single PCR specific primer, five µg EcoRI and KpnI were used to digest the genomic DNA before ligating it into a strategene plasmid vector pBlueScript. Dual PCR analysis  – using the RARA genespecific primer and then the vector-specific M13 reverse primer was done to strengthen the genomic region. The resulting PCR product was replicated into an Invitrogen and the sequence analyzed.

Concerning the results, the authors discovered that RARA intron 2 approximated at 17 kb with 3 KpnI. A peculiar 5b band was discovered via Southern blot scrutiny. EcoRI analysis located the breakpoint in KpnIeEcoRI fragment directly 5’ towards probe 2 southern blot testing located the breakpoint in the 1-kb KpnIeEcoRI section. All in all, PML sequence was shown to be the anonymous partner.

Ultimately, to show the direction in which further research should take ,the authors explain that they have not  conducted their sequence examination with regard to indentifying the chromosome 3 locus within which the enigmatic PML-RARA was introduced. Moreover, after mentioning that huge genomic probes have certain limitations, the authors point out that new karyoptic leukemic abnormalities should be subjected to further analysis.

Reference

1.      Chattopadhyay A,  Redner R. L. Cryptic insertion of PML-RARA into the 3p25 locus in an acute promyelocytic leukemia with t(3;17)(p25;q21). Cancer Genetics and Cytogenetics 2010; 201 (2010): 28-31.

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