Genotyping RFLP

Indirect methods for genotyping, RFLP, primer-extension methods (e.g., Snapshot, Applied Biosystems Inc. (ABI), matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), primer extension method to assay allele-... 

Wicklow, D. T., McAlpin, C. E., Yeoh, Q. L. (2007). Diversity of Aspergillus oryzae genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia. Mycoscience, 48, 373-380. 

Mycobacteria of the Mycobacterium avium complex are implicated in disseminated bacterial infections in AIDS patients. RFLP studies followed by hybridization with radiolabeled probe specific for an insertion sequence in M. avium (IS 1311) have been useful for typing M. avium stains (R2). A variety of molecular techniques are available for the diagnosis of Chlamydia trachomatis infection. In addition to PCR, a method based on the ligase chain reaction has also been found to be sensitive to the detection of C. trachomatis infection in urine specimens collected from male and female subjects (VI). The differentiation between low-risk genotypes of human papilloma virus (HPV 6 or 11) from genotypes of high... 

The gene frequency measures the proportion of chromosomes that contain a specific allele. To continue the RFLP example given above, we wish to estimate the frequencies of alleles 1 and 2 in our population. Each individual with the 1-1 genotype has two copies of allele 1, and each heterozygote (1-2 genotype) has one copy of allele 1. Because each diploid somatic ceU contains... 

Venous blood (8 mL) was collected into 15-mL tubes containing 50 mmol/L disodium EDTA, and genomic DNA was extracted by the standard (phenol/chloroform) method. The genotype frequencies for GSTPl were determined by polymerase chain reaction (PCR)/RFLP-based methods using DNA extracted from peripheral lymphocytes. 

Methods for identification of human protozoan parasites in the tissue of molluscan shellfish include (1) immunofluorescent antibodies (IFA) alone or in combination with immunomagnetic separation (IMS), (2) polymerase chain reaction (PCR) alone or combined with restricted fragment length polymorphism (RFLP) for genotyping, (3) multiplexed nested PCR, and (4) fluorescence in situ hybridization (FISH). Infectivity of the parasites recovered from the shellfish are usually assessed by mouse bioassays. 

The main advantages of RFLP analysis are that it is simple to develop and use. It also does not require expensive equipment, and it is effective for genotypingasmall number of samples. The principle disadvantages are that it is time consuming, labor intensive, and not amenable for large numbers of samples or genotypes. Another drawback is that not all SNPs produce a usable restriction site. 

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