Pre-hybridize the membrane: Pre-hybridize the membrane by incubating it in a solution that contains blocking agents, such as salmon sperm DNA or blocking reagents.This process ensures that the DNA strands become single-stranded, allowing the probe to bind to the complementary DNA sequences. Denature the DNA on the membrane: Denature the DNA on the membrane by incubating it in a denaturing solution.This solution will be used to hybridize with the DNA on the membrane and detect specific DNA sequences. Prepare the hybridization solution: Prepare a hybridization solution that contains the DNA probe and a buffer solution.Ensure you have the Southern blot membrane ready. Obtain the Southern blot membrane: Southern blots involve transferring DNA fragments from a gel onto a membrane, which is then used for DNA hybridization.To read Southern blots, which are used in molecular biology to detect specific DNA sequences, follow these steps: DNA should be digested with a second enzyme to resolve comigrations.Ĭontinue reading here: Introduction 11 Overview Restriction enzymes that produce large germline fragments are more likely to comigrate with nongermline bands. Comigration of rearranged bands with germline bands can be a major problem when using the restriction enzyme HindIII in combination with the constant region probe for the TCRp chain. Comigration occurs when both the germline band and the rearranged band migrate to the same point in the gel. This will eliminate misinterpretation of uncommon RFLPs or partial digests as a clonal rearrangement.Ĥ. If results from the two digests are not consistent, a third digest should be performed. It is recommended that two enzymes be used in combination with each probe. Thus, laboratories must be aware of RFLPs generated by routinely used restriction enzymes and DNA probes. Restriction fragment length polymorphisms (RFLPs) and partially digested DNA may produce nongermline bands that are not owing to rearranged antigen receptor genes. Abbreviations: MW = molecular weight marker Pat = patient sample.ģ. The molecular findings in conjunction with morphology are suggestive of a posttransplant lymphoproliferative disorder. The PCR gel demonstrates the presence of EBV DNA, and the Southern blot confirms the clonal nature of the EBV DNA. The patient now presents with a lung mass suspicious for lymphoma. Status post autologous bone marrow transplant for Hodgkin disease of a 39-yr-old female. It is important to be aware of these conditions to avoid erroneous diagnoses of malignancy.įig. Several "benign" clonal lymphoproliferative conditions exist and include immunodeficiency settings associated with Wiscott-Aldrich syndrome and autoimmune deficiency syndrome, angioimmunoblastic lymphadenopathy, posttransplant immunosuppression, congenital immunodeficiency, and benign monoclonal gammopathy. The use of multiple enzyme-probe combinations helps overcome this problem.Ģ. Additionally, certain rearrangements may produce very large restriction fragments, and hence the DNA may transfer poorly, causing these rearrangements to be missed. One usually sees an 8.5-kb band in addition to the usual 11- and 4-kb germline bands, which is owing to a relatively resistant EcoRI site (3,21). A common example of partial digestion is EcoRI-digested DNA hybridized with a TCRß probe. False-positive results may also arise from either a partial digestion or gene polymorphisms. False-negative results with Southern blotting usually occur because the clonal population is below the sensitivity level of Southern analysis or because of tissue-sampling error. The following guidelines are useful for interpretation of Southern blots:ġ. Abbreviations: MW = molecular weight marker Pat = patient sample.Įach rearranged gene and is likely to represent more than one clone. PCR gel and slot blot in a patient with mantle cell lymphoma, demonstrating the classic bcl1 gene (t ) rearrangement. A marked difference in intensity of the two bands implies different dosages of Fig. Two rearranged bands in a single lane hybridized with one gene probe may be a result of two coexisting clones within the same tissue or of rearrangements of both alleles for that specific gene (25). Generally, a positive result will be seen with both enzymes and is usually also positive by PCR. The intensity of the rearranged band is proportional to the percentage of clonal cells in the sample being tested. Any rearrangement of the Ig or TCR genes will appear as a band with variable intensity and a size different from the germline band. The presence of germline bands is interpreted only as a negative result.
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