Lecture 25

DNA Diagnostics

Interrogation of DNA sequence, without going to the trouble of sequencing!


Overview

We've had a fair opportunity to discuss DNA diagnostic methods in this course, so much of this may seem like "old hat" to you. For example in the laboratory, you used restriction enzymes to study the structure of a plasmid you had constructed. We had made predictions of the restriction map using a computer program, and you checked the structure by experiment - that was a diagnostic test.

Often times people develop specific tests to look at just a few nucleotides (for example a restriction site) because that is easier than sequencing the DNA. Here are some examples:

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

RFLP is useful for DNA typing, and an application of its use for typing of pine trees is underway at U.C. Berkeley.

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.

Heteroduplex gel mobility shift

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:


 

Method: Denaturing Gradient Gel Electrophoresis (DGGE)

update July 1, 1990

C. Helms

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.

http://hdklab.wustl.edu/lab_manual/dgge/dgge1.html

DGGE kit, ready to buy from C.B.S. Scientific Co. Inc

An example of DGGE (and a question about GC clamps)

Why do clamped primers give us smears instead of bands?

E-beta primers:

non-clamped
Forward Primer - GAG GGA CTC GGG CAT CTT
Reverse Primer - CAC CCC GTC CTG TCT CAC

clamped
Formard Primer - CGC CCG CCG CGC CCC GCG CCC GCC CCG CCG CCC CCG CCG CGA GGG ACT CGG GCA TCT T
Reverse Primer - CAC CCC GTC CTG TCT CAC

DGGE gel results:
30-70% denaturant concentration DGGE gel
Run for 5 hrs at 275 volts in 1X TAE
The first 4 lanes were amplified with the clamped primer.
The second 4 lanes were amplified with the non-clamped primer.

SSCP

SSCP = Single Strand Conformational Polymorphism, and this is a slightly different approach to finding changes in DNA sequence. A PCR product is denatured, and the separated strands are allowed to form a natural secondary structure. The structure adopted is highly dependent on sequence, and single nucleotide changes can often be recognized by the change that they cause in the conformation and gel mobility.



Sequence variability in p27 gene of Citrus Tristeza Virus (CTV) revealed by SSCP analysis

Gago-Zachert, et al.

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."

http://ejb.ucv.cl/content/vol2/issue1/full/3/index.html

  Here is another example of an application - this one a bit more flavorful:

Relationship of a PCR-SSCP at the Bovine Calpastatin Locus with Calpastatin Activity and Meat Tenderness

Chung, et al.

"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."

http://ohioline.ag.ohio-state.edu/sc170/sc170_3.html

CFLP

CFLP = Cleavase Fragment Length Polymorphism is an approach that allows fixation of differences in conformation by sensitivity to an endonuclease.

CFLP Scan(tm) and CFLP Scan(tm)-XL Kits
A fast, sensitive alternative to SSCP and RFLP


Submitted by: Mary Ann D. Brow, Mary Oldenburg, Victor Lyamichev, Laura Heisler, Jeff Grotelueschen, Natasha
Lyamichev, Sergei Kozyavkin, Lance Fors, James Dahlberg, Lloyd Smith, and D. Michael Olive*
Third Wave Technologies, Inc.
Madison, Wisconsin 53711
*Corresponding author: Email: MOLIVE@TWT.COM

During the studies that led to the development of single-stranded conformational polymorphism (SSCP) assays, Hyashi (1) noted that, following denaturation, DNA strands assume folded hairpin-like structures that are nucleotide sequence dependent. We have recently identified a family of endonucleases that can recognize and cleave these folded structures at the junctions between the single-stranded and duplexed regions.

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.