The technique for RFLP analysis is, however, slow and cumbersome. It requires a large amount of sample DNA, and the combined process of probe labeling, DNA fragmentation, electrophoresis, blotting, hybridization, washing, and autoradiography can take up to a month to complete. A limited version of the RFLP method that used oligonucleotide probes was reported in 1985. The results of the Human Genome Project have largely replaced the need for RFLP mapping, and the identification of many single-nucleotide polymorphisms (SNPs) in that project (as well as the direct identification of many disease genes and mutations) has replaced the need for RFLP disease linkage analysis (see SNP genotyping). The analysis of VNTR alleles continues, but is now usually performed by polymerase chain reaction (PCR) methods. For example, the standard protocols for DNA fingerprinting involve PCR analysis of panels of more than a dozen VNTRs.
RFLP is still used in marker-assisted selection. Terminal restriction fragment length polymorphism (TRFLP or sometimes T-RFLP) is a technique initially developed for characterizing bacterial communities in mixed-species samples. The technique has also been applied to other groups including soil fungi. TRFLP works by PCR amplification of DNA using primer pairs that have been labeled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer. The results are analyzed either by simply counting and comparing bands or peaks in the TRFLP profile, or by matching bands from one or more TRFLP runs to a database of known species. A number of different software tools have been developed to automate the process of band matching, comparison and data basing of TRFLP profiles.
The technique is similar in some aspects to temperature gradient or denaturing gradient gel electrophoresis (TGGE and DGGE).
The sequence changes directly involved with an RFLP can also be analyzed more quickly by PCR. Amplification can be directed across the altered restriction site, and the products digested with the restriction enzyme. This method has been called Cleaved Amplified Polymorphic Sequence (CAPS). Alternatively, the amplified segment can be analyzed by allele-specific oligonucleotide (ASO) probes, a process that can often be done by a simple dot blot.