Riff-lips
RFLP = restriction fragment length polymorphism. This is simply asking the question of whether specific restriction sites are present in a genomic DNA region. For example, the following image outlines the method of detecting a missing site (for Mst II) in hemoglobin S:
While RFLP can be combined with southern analysis using a labeled probe, it is also
frequently combined with polymerase chain reaction so that the results can be studied
on a standard gel.
A small segment of a genome is amplifed by PCR, then tested with a restriction enzyme to see if a site (or sites) are present. If the DNA digests, what do you know? Only that the restriction enzyme recognition sequence was present, and this amounts to 4 to 6 nucleotides of information. If the DNA does not digest, what do you know? Only that one or more of the 4 to 6 nucleotides of information in the putative site is mutated or absent. A single nucleotide change can eliminate a restriction site.
Here is an example of an RFLP experiment:
You can see that there is a banding pattern difference between two species of plant parasitic nematode. The method of typing works best with closely-related taxa.
| Species | Fragments (bp) |
| H. glycines (SCN) | 252, 89 |
| H. schactii (SBCN) | 181, 89, 58 |
Other PCR methods
I think you can already imagine how one could use PCR to test for a specific nucleotide. If a primer ends on a polymorphic nucleotide at its 3' end, it cannot be extended unless it matches. Using an enzyme such as Taq polymerase, which cannot correct such a mismatch, the presence or absence of extension products can be diagnostic.
Sequence anomalies such as mini and micro-satellites are readily detected by polymerase
chain reaction. One example of this diagnostic is for "variable numbers of tandem
repeat" regions or VNTRs. Primers to unique regions outside of a repeat structure
are used to generate fragments that contain the complete set of repeats. Different
individuals may have different alleles of the region, having more or fewer copies
of the repeat, and this shows up as difference in fragment size after PCR.
Detection of microsatellites, usually tri or tetra-nucleotide repeats, are also statistically powerful ways of typing a genome.
We recall from our earlier discussion of gel electrophoresis, that DNA fragments migrate on a gel partly as a function of length but also as a function of "shape". Distortions of structure can lead to alterations in mobility, and this is the basis for a group of methods used in DNA diagnosis.
This tends to work best with short fragments, so scanning long segments of DNA for changes takes some effort. The overall approach can be made more sensitive by running the gel as a "denaturing gradient", made for example with a gradient of urea:
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Method: Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gels are used to detect non-RFLP polymorphisms. The small (200-700 bp) genomic restriction fragments are run on a low to high denaturant gradient acrylamide gel; initially the fragments move according to molecular weight, but as they progress into higher denaturing conditions, each (depending on its sequence composition) reaches a point where the DNA begins to melt. The partial melting severely retards the progress of the molecule in the gel, and a mobility shift is observed. It is the mobility shift which can differ for slightly different sequences (depending on the sequence, as little as a single bp change can cause a mobility shift). Alleles are detected by differences in mobility. |
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An example of DGGE (and a question about GC clamps) Why do clamped primers give us smears instead of bands?
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SSCP
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Sequence variability in p27 gene of Citrus Tristeza Virus (CTV)
revealed by SSCP analysis |
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| Abstract "Citrus tristeza closterovirus (CTV), is a phloem-limited virus transmitted by aphids in a semipersistent manner. The genome of CTV is composed of a ssRNA with two capsid proteins: CP, covering about 95% of the particle length, and a diverged coat protein (dCP), present only in one end of the particle, forming a rattlesnake structure. dCP is the product of p27 gene for which it is also postulated a function in the transmissibility by aphid vectors. Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of single-strand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolate reducing considerably the amount of nucleic acid sequenciation necessary to gain that knowledge." |
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Relationship of a PCR-SSCP at the Bovine Calpastatin Locus with
Calpastatin Activity and Meat Tenderness |
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| "This study was designed to investigate the effects of calpastatin activity and myofibril fragmentation index on meat tenderness and the effects of calpastatin genotypes determined using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) analysis on these variables." |   |
CFLP
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CFLP
Scan(tm) and CFLP Scan(tm)-XL Kits
reference: http://www.biochem.boehringer-mannheim.com/techserv/CLEAVASN.HTM
Figure 1: Schematic illustration of the principle underlying the CFLP assay. Wild type and mutant DNA fragments differing by as little as a single nucleotide change are denatured by heating and then cooled to a predetermined temperature optimum, allowing the formation of sequence-dependent secondary structures. Upon reaction with Cleavase I, these structures are cleaved, and the resulting fragments resolved by electrophoresis. In the ?bar code? pattern that is obtained, mutational differences are reflected as the appearance or disappearance of bands, band shifts, or changes in band intensity near the site of the actual mutation. |
Molecular Beacons
Exposes fluorescence when opened:
Related methods:
TaqMan
PCR-forced opening of an oligonucleotide.