While highly discriminatory, RFLP analysis of VNTRs has several drawbacks, including:

• The process is extremely laborious and time-consuming
• Radioactive probes pose health and disposal risks (although chemiluminescent technology eliminated this risk)
• A relatively large amount of sample is required to perform the tests
• The method requires high molecular weight, un-degraded DNA
• The use of yield gels is an essential, but time consuming, step in the analysis not only to estimate the amount of DNA recovered but also to determine the suitability of the sample for analysis

Read more about yield gels in course: DNA Extraction & Quantitation

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RFLP allele assignment was performed by incorporating a reference sizing ladder in the electrophoresis and comparing the fragments to the size markers.  Various approaches were developed to deal with the intrinsic variation in the method, including the use of windows or bins to express the range within which the true fragment size lay.  Apart from the undesirability of having to explain why there was not a definitive size assigned to a fragment, the inherent continuous distribution of allelic states posed challenges for the national DNA database software (CODIS).

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