
	1999 Summer Scholars Program

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A New DNA Diagnostic Test based on Fluorescence Polarization Detection

By By Sherif Latif
Mentor: Pui-Yan Kwok MD, Ph.D.
Department of Genetics, Washington University School of Medicine, St. Louis, MO

Point mutations, or Single Nucleotide Polymorphisms (SNPs), are becoming a frequently utilized tool in detecting genetic disease genes, such as the BRCA1 breast cancer gene. SNPs are useful because they occur very frequently in the DNA sequence, and thus also occur in critical areas, directly affecting protein structure or expression levels. One successful method for detecting SNPs, the TaqMan assay, utilizes the 5'-nuclease activity of Taq DNA Polymerase to digest a probe labeled with both a fluorescence and a quencher molecule. When the quencher molecule is attached to the fluorescence molecule, the quencher molecule will absorb all of the fluorescence. By designing the probe with the desired sequence, i.e. complementary strand of the polymorphism, it then becomes relatively easy to test for the polymorphism itself.

This process begins when DNA is introduced to a mix containing Amplitaq Gold Polymerase, the molecule that will duplicate the DNA during the PCR process, and the probe. During PCR the probe will bind to a specific site which contains its complementary sequence. The Amplitaq Gold Polymerase will then come and cleave the probe, such that the fluorescence molecule separates from the quencher molecule. This will then allow fluorescence to be detected using the 7700 Sequencer. If, on the other hand, the complementary strand containing the polymorphism does not exist in the DNA sample, then the probe will not bind. Thus, when the Amplitaq Gold Polymerase replicates the DNA sample, the probe will remain intact with the quencher molecule absorbing the fluorescence.

Several problems arise, however. Because the probe contains both a fluorescence and a quencher molecule, the overall price of the probe increases dramatically. Therefore, any large-scale use of the TaqMan assay is rather expensive. In addition, the ability to multiplex is limited.

These deficiencies are resolved using another method known as Fluorescence Polarization (FP). FP is based on the observation that when a fluorescent molecule is excited by plane-polarized light, it emits polarized fluorescent light into a fixed plane if the molecules remain stationary between excitation and emission. Because the molecule rotates and tumbles in space, FP is not fully detected by an external detector. The FP of a molecule is proportional to the molecule's rotational relaxation time (the time it takes to rotate through an angle of 68. 5°), which is related to the viscosity of the solvent, absolute temperature, molecular volume, and the gas constant. Therefore, if the viscosity and temperature are held constant, FP is directly proportional to the molecular volume, which is directly proportional to molecular weight. If the fluorescent molecule is large (with high molecular weight), it rotates and tumbles more slowly in space and FP is preserved. If the molecule is small (with low molecular weight), it rotates and tumbles faster and FP is largely lost or depolarized.

In each experiment there are four possible outcomes for the type of genotype that can occur. We can either receive a homozygote for the first allele, a homozygote for the second allele, a heterzygote containing the first and second alleles, or we can get a negative. Using two different fluorescence molecules, we received four different data sets that we could correlate to the TaqMan Assay. The results obtained prove that FP is an excellent companion to the TaqMan assay, and thus can help detect genetic mutations.

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