Bioethics and the Impact of Human Genome Research in the 21st Century

4.1. DNA Polymorphism Technologies
pp. 35-39 in Bioethics and the Impact of Human Genome Research in the 21st Century

Author: Sumio Sugano (Tokyo University Medical School)

Editors: Norio Fujiki, Masakatu Sudo, and Darryl R. J. Macer
Eubios Ethics Institute

Copyright 2001, Eubios Ethics Institute All commercial rights reserved. This publication may be reproduced for limited educational or academic use, however please enquire with the author.

Introduction
Just as no one has the same face, so the sequence of each individual's genome is subtly different. These individual genetic variations are known as polymorphisms. The following types of DNA polymorphism due to differences in base sequence are known,

single nucleotide polymorphisms SNP

insertions and deletions

variations in the number of repeat sequences (microsatellites etc.) etc.

Of these the most common, and those thought to be the most useful in searching for genes related to multi-factorial diseases such as diabetes and hypertension, are SNPs. It is said that in the human genome contains one SNP can be found every 4-700 bases. In fact, as sequencing of the human genome has progressed the presence of a multitude of SNPs, maybe 1-2 million, is already becoming clear. To utilize these SNPs in the search for disease-related genes requires their identification in the genomes of patient and control groups of several thousand people, and the correlation of these with the illness concerned. Therefore a method to determine SNPs quickly and cheaply is needed. Here I'll summarize the methods used in SNP-typing.

The Principles of SNP-typing
Various methods for SNP-typing have been proposed. When discussing their relative merits it is necessary to analyze the individual steps of each typing system, and weigh their advantages and disadvantages. Firstly we must consider the principles of SNP identification. To identify an SNP we must first identify directly or indirectly the base involved, but there are not many methods for this; 1) those using sequence reaction 2) those using hybridization 3) methods using neither. Only the SSCP method falls in this last category, so I'll leave its details to the next section and discuss the first two here.

In the first a primer is set up near the SNP and a normal sequence reaction including dideoxynucleotides is performed (Fig 1). The result is judged by determining which base is introduced in the position of the SNP. Representative is the single base-extension method (Fig 1a). A primer for the section immediately before the SNP is synthesized, and a chain-extension reaction using only dideoxynucleotides is performed. Then the type of base incorporated in the SNP position is determined. Alternatively in the probe method by using some of the nucleotides as their normal deoxy, and not as the dideoxy form, the SNP can be detected by the variation in length of the product (Fig 1b). In the hybridization method an oligonucleotide is prepared to match each SNP sequence and the one binding is detected. There are three methods utilizing this principle, the sequence by hybridization method (Fig 2), dye-labeled oligonucleotide method (Fig 3) and the invader method (Fig 4).

Detection Methods
Whichever of the above principles we might use to identify and determine SNPs, in the end we have to present results in a form visible to the human eye. To this end various detection methods are used. Those being researched at present include the DNA chip, mass analysis and fluorometers.

There are two methods using DNA chips. In one single base extension is performed and the products obtained are hybridized on a DNA chip, which is then read. This has been commercialized by Affimetrix as the Genflex tag array. In the other the portion containing the SNP is amplified using PCR, and the product hybridized on the chip and its sequence read, in the end this is sequence by hybridization. This requires a new DNA chip to be made for eqachSNP to be detected, and while there are Affimetrix Genechips for p53 mutations and analysis of CYP450 poylmorphisms, problems of cost mean at present this method is not generally used. Should important SNPs be identified in the future, it may well come into its own in routine clinical testing.

SEQUENOM's MassArray method is famous technique using TOFMS. SNPs are expressed using the probe method as oligonucleotides of varying length, and MALDI-TOFMS is used to determine the length (mass) of these. A characteristic of the method is that the measurement time per sample for TOFMS is short, so many samples can be dealt handled. On the other hand all the high molecular weight ions in the sample are detected, so unless the sample is properly purified the background noise is high, and good results are unobtainable.

Figure 1: SNP-typing by sequence reaction

A: Single base-extension method. When the product obtained by this method is directly analyzed by sequencer it s known as SnaPshot.

B: Probe method.

Figure 2: Simple hybridization method

The diagram is drawn as if the genome DNA sequence had been determined, but usually in sequence by hybridization while being amplified the portion containing the SNP is fluorescence-labeled, and the probe oligonucleotide is immobilized on a base in the form of a DNA chip.

Figure 3: Dye-labeled oligonucleotide ligation method

This method works on the principle of mismatch detection. When ligation is performed using a ligase from e.g. E.coli then should there be a mismatch in one of the fragments the reaction doesn't proceed well. In practice a ligase from heat-resistant bacteria is used.

Figure 4: Invader method

This method uses an oligonucleotide, the signal-probe, that can hybridize with the right-hand side, SNP-containing section and that contains a sequence unrelated to the SNP region called the "flap-part", and an oligonucleotide, the invader-probe, that can hybridize with the left-hand side, the 3' terminal of which exactly overlaps with the SNP, but for which any base may be used. When hybridization is successful the signal-probe hybridizes with the right-hand side with the 3' terminal of the invader-probe sometimes obstructing the binding. An enzyme known as a cleavase recognizes this structure and cleaves off the flap-part. When the SNP is a mismatch then the recognition site cannot form. The flap-part separated by the cleavase then acts as an invader-probe towards a fluorescent probe and a flap-recognizing oligonucleotide labeled with a quenching dye, forming a recognition site for the cleavase. As a result a cleavase reaction occurs, the fluorescent dye is released, and fluorescence can be detected.

Figure 5: Taqman method

In this method the characteristic that only a hybridized oligonucleotide is cleaved during the PCR reaction is used to detect whether or not the oligonuleotide was hybridized. The probe contains a fluorescent dye and a quenching dye bound at a suitable distance so that there is no fluorescence. When the oligonucleotide is cleaved the dyes are separated and fluorescence is seen.

Figure 6: Molecular beacon method

This method utilizes the fact that while when the fluorescent dye and the quenching dye are a suitable distance apart no fluorescence occurs, on hybridization that distance increases and fluorescence can occur to detect whether or not hybridization has occurred.

Among the methods using fluorometry are the Taqman method (Fig 5), the molecular beacon method (Fig 6) and the invader method. As shown in the diagram, in the Taqman and molecular beacon methods a special probe is prepared, designed to fluoresce on hybridization. The target SNP region is amplified using PCR and when hybridization occurs is detected. In practice, along with PCR reagents the Taqman probe or molecular beacon is introduced into the reaction mixture, and whether or not fluorescence becomes stronger as the PCR proceeds is measured. In the invader method whether or not an oligonucleotide known as a signal-probe hybridizes with the SNP is examined. The characteristic of this method is the use of another oligonucleotide known as the invader-probe along with the signal-probe. Along with the genome DNA these form a triple-strand which is recognized by an enzyme known as a cleavase, which then cuts and separates the upper, flap part of the signal-probe. This flap is then measured.

Among the methods relying on fluorescence detection methods is that using Luminex beads. These are polystyrene beads containing orange and red fluorescent material in differing amounts. By measuring the relative strengths of their fluorescence they can be separated by a flow-cytometer. Various oligonucleotides are bound to the beads, and by hybridizing fluorescent oligonucleotides formed by the dye-labeled oligonucleotide ligation method to these beads it is possible to analyze simultaneously 64 types of SNP.

Multiplex PCR
It is thought that investigation of disease-related genes using SNPs will require the analysis of many tens of thousands. A single human genome DNA is a vast super molecule made of three billion base pairs. To detect by the above methods the SNPs existing in one small part of this vast molecule requires at least 1-3 thousand template genome DNAs, or 3-10ng. To analyze ten thousand SNP positions individually by PCR would require 30-100ng of genome DNA.

If a number of SNPs could be detected not one by one but at the same time then the amount of genome DNA required should be greatly lessened. For example, if 10 SNP positions could be detected at once, then only a tenth the amount of genome DNA would be needed for polymorphism analysis etc. The technology for achieving this is multiplex DNA. At present 50-100 positions can be simultaneously analyzed by PCR. Furthermore methodology for amplifying the whole DNA by using e.g. random hexamer etc are under investigation.

Conclusion
We have galloped through a look at some SNP-typing methods. There are various methods, but at present we can't say which will prove best. Moreover, depending on ones aims requirements will differ. For example, in investigations on disease-related genes the researcher needs to be able to choose fairly freely a large number of SNPs, while for testing a small number of SNPs in a large number of samples need to be measured cheaply. The optimum technology for each won't be the same. From here on we need to pursue development targeted to use, and cost-reduction.

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