RFLPs analysis

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. 

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. 

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

The accurate analysis of genetic variation in nematodes has major implications for parasite identification and for investigating population genetic structures (Lejambre, 1993 Grant, 1994). Conventional DNA methods have been valuable and their application has provided a wealth of molecular data (reviewed by McManus and Bowles, 1996), but little attention has been paid to their capacity to resolve sequence variation (reviewed by Gasser, 1997). For instance, PCR-RFLP analysis using (multiple) restriction endonucleases is useful, but sequence variation may go undetected as... 

RFLP analysis by the Southern blot procedure and detection with probe specific for each VNTR are the basis of DNA fingerprinting. Using just four DNA probes, paternity can be excluded with a cumulative probability of 99.9% (J2). 

The initial goal is to identify an RFLP that is dosefy linked to the gene involved in the disease. Once such markers have been identified, Southern blots may be used to predict which members within a mily have inherited the allele assodated with the disease. An example of an RFLP analysis based on a mutation in a restriction site is presented here. 

Figure 1-7-10. EeoRI RFLP Analysis of a Family With Myotonic Dystrophy...

RFLP analysis of the p-globin gene for genetic testing has been replaced, by PCR In combination with ASO probes on dot blots. The blot shown here corresponds to the family whose pedigree is shown in Figure T7-9. In the mutant allele, glutamate (E) at codon 6 is replaced by valine (V). 

Southern blot DNA restriction fragments (RFLP analysis)... 

Correct answer = A. ELISA assays are commonly used to detect production of proteins. Northern blots could detect mRNA production, but would riot guarantee that the protein was made. Southern blots, ASO probes, arid RFLP analysis could be used to study the DNA inserted into the goat, but would not give information about protein production. 

As the genes themselves are identified and sequenced, so the need for RFLP markers declines since specific DNA probes (see Topic 12) for the most common types of gene defect can be employed. In addition, the use of the polymerase chain reaction (PCR see Topic 16) in screening for human genetic disease is becoming the method of choice rather than RFLP analysis since it is much faster to perform and requires far less clinical material for analysis. 

Osborn, A. M., Moore, E. R. B., and Timmis, K. N. (2000). An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2,39-50. 

Warren JT Jr, Peacock ML, Fink JK. An Rsal polymorphism in the human serotonin receptor gene (HTR1 A) detection by DGGE and RFLP analysis. Hum Mol Genet 1992 1 778. 

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