Leonard Klevan

[66] Biotinylated nucleotides for labeling and detecting DNA

Publisher Summary The sensitivity, selectivity, and ease of use of nucleic acid probes and the availability of efficient protocols for the isolation and cloning of specific DNA sequences have led to the development of a wide selection of probes for biomedical and clinical applications. In the basic hybridization protocol, a labeled nucleic acid probe is annealed to a complementary DNA or RNA target sequence, which either is in solution or is immobilized on an inert support. The labeled nucleic acid probe is used to determine the presence or absence of the target sequence in the reaction mixture. An effective nonradioactive detection system should recognize the annealed nucleic acid probe with a degree of precision comparable to that obtained in the primary hybridization reaction. The presence of either a free amine or biotin may also be ascertained by chemical analysis. Compounds containing primary amines give positive color development when spotted on thin layer chromatography (TLC) plates and sprayed with ninhydrin. The first approach uses iminobiotin, an analog of biotin in which the ureido group has been replaced with a guanido group. Iminobiotin binds to avidin with a binding constant that increases with increasing pH and nonprotonated iminobiotin binds efficiently to avidin.

RFLP Typing: A New Highly Polymorphic VNTR Locus and Chemiluminescent Detection

There are two basic DNA typing methodologies available to the forensic scientist for characterizing biological evidence. The first technology to gain wide use in the forensic arena was typing of DNA for variable number of tandem repeat (VNTR) loci by restriction fragment length polymorphism (RFLP) analysis. RFLP typing is well-defined, provides a high degree of discrimination, and can be accomplished, at times, with less than 50 ng of high molecular weight genomic DNA. This methodology has been validated for forensic applications (Budowle and Baechtel 1990). The other strategy for typing DNA is based on increasing the number of copies of a target sequence of DNA by amplification using the polymerase chain reaction (PCR) (Saiki, et al. 1985). Since the number of target sequences of interest is increased dramatically by PCR, simplified typing methods can be used for determining DNA polymorphisms in a sample. The advantages a PCR-based technology offers, compared with the currently employed RFLP approach, are: 1) augmented sensitivity and specificity, 2) decreased assay time and labor, 3) absence of an isotopic label, and 4) many degraded DNA samples can be amplified by PCR and subsequently typed because alleles amenable to PCR are much smaller in size compared with alleles detected by RFLP analysis. These qualities combine to make PCRbased technologies extremely useful tools for analyzing biological material found at crime scenes.