RFLPs

Goate, A.M., Cooper, D.N., Hall, C., Leung, T.K., Solomon, E., Li, L. (1987). Localization of a human heat shock hsp70 gene sequence to chromosome 6 and detection of two other loci by somatic cell hybrid and RFLP analysis. Hum. Gen. 75, 123-128. 

The detection of restriction fi agment length polymorphisms (RFLPs) facilitates prenatal detection of hereditary disorders such as sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington s disease. Detection of RFLPs involves cleavage of double-stranded DNA by restriction endonucleases, which can detect subtle alterations in DNA that affect their recognized sites. Chapter 40 provides further details concerning the use of PCR and restriction enzymes for diagnosis. 

Short (2-6 bp), inherited, tandem repeat units of DNA occur about 50,000-100,000 times in the human genome (Chapter 36). Because they occur more frequently—and in view of the routine application of sensitive PCR methods—they are replacing RFLPs as the marker loci for various genome searches. 

Variable numbers of randomly repeated (VNTR) units are one common type of insertion that results in an RFLP. The VNTRs can be inherited, in which case they are useful in estabhshing genetic association with a disease in a family or kindred or they can be unique to an individual and thus serve as a molecular fingerprint of that person. 

Exonuclease An enzyme that cleaves nucleotides from either the 3 or 5 ends of DNA or RNA. Fingerprinting The use of RFLPs or repeat sequence DNA to establish a unique pattern of DNA fragments for an individual. 

Use of probes to define marker loci Probes identify STSs, RFLPs, SNPs, etc thousands, covering all the chromosomes, are now available. It is desirable to flank the gene on both sides, clearly delineating it. 

Abbreviations STS, sequence tagged site RFLP, restriction fragment iinked poiymorphism SNP, singie nucieotide poiymorphism YAC, yeast artificiai chromosome BAC, bacteriai artificiai chromosome PCR, poiymerase chain reaction. 

The first human DNA SRM developed by NIST was designed in the late 1980 s to standardize Restriction Fragment Length Polymorphism (RFLP) procedures which at that time were very new developments in the application of DNA to forensic analysis. 

SRM 2390 can be used in several different ways depending on quality assurance requirements. Components of the SRM are designed to provide assurance that each step of the RFLP protocol is functioning properly, but they can also be used for trouble shooting, for calibrating equipment, and for testing the efficacy of new lots of reagents. 

Eisenberg AJ, Gibson P, Nandi S and Wang L (1991) The development and implementation of a HAE Ill-based RFLP system for parentage testing in Texas. Proceedings from the Second International Symposium on Human Identification, Promega Corporation, pp 163-180. 

These DNA markers have been successfully employed to track specific strain-associated loci in endo- and ectomycorrhizal populations from agricultural land, forest nurseries, plantations, and natural ecosystems. The simplest strategy (digesting PCR-amplified ITS with selected endonucleases) has identified their symbionts in various ecosystems (18,36-38). Species discrimination by ITS-RFLP matching can be improved by comparing data for the targeted DNA with those on sequence databases (37). 

Smeraldi, E., Zanardi, R. et al. (1998). Polymorphism within the promoter of the serotonin transporter gene and antidepressant efficacy of fluvoxamine. Mol. Psychiatry, 3(6), 508-11. Wang, S. L., Huang, J. D. et al. (1993). Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects polymorphism in RFLP and DNA sequence of CYP2D6. Clin. Pharmacol. Ther., 53(4), 410-18. 

RFLPs are often a reflection of individual genetic diversity and are not related to a clinical phenotype, but occasionally they can be diagnostic of an inherited disease. This technique is relatively new yet, it has been applied to the prenatal detection of sickle cell anemia, thalassemia, phenylketonuria, a,-antitrypsin deficiency, Huntington s chorea, Duchenne muscular dystrophy, hemophilia A and B, cystic fibrosis, and several other, diseases. 

Reverse genetics has been applied to diseases such as Duchenne muscular dystrophy and cystic fibrosis, in which the responsible enzymes are unknown and the disease results from a significant deletion. By combining RFLP analysis with cytogenetics, it has been possible to increasingly narrow the location of the defective genes to small regions on the affected chromosomes. 

Iwahori, H., Tsuda, K., Kanzaki, N., Izui, K. and Futai, K. (1998) PCR-RFLP and sequencing analysis of ribosomal DNA of Bursaphelenchus nematodes related to pine wilt disease. Fundamental and Applied Nematology 21, 655-666. 

Romstad, A., Gasser, R.B., Nansen, P., Polderman, A.M., Monti, J.R. and Chilton, N.B. (1997b) Characterization of Oesophagostomum bifurcum and Necator americanus by PCR-RFLP of rDNA. Journal of Parasitology 83, 963-966. 

PCR-linked restriction fragment length polymorphism (PCR-RFLP) analysis... 

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