For citation purposes: Hartwig FP. A probe-based alternative method for allele-specific amplification for targeted haplotyping. OA Molecular Oncology 2013 Mar 01;1(1):3.

Hypothesis

 
Cancer Genetics & Genomic Instability

A probe-based alternative method for allele-specific amplification for targeted haplotyping

FP Hartwig*
 

Authors affiliations

Federal University of Pelotas Oncology Research Group, Technology Development Centre (Biotechnology Unit), Federal University of Pelotas, Pelotas, Brazil

* Corresponding author Email: fernandophartwig@gmail.com

Abstract

Introduction

Genetic factors play important roles in cancer and other complex diseases. An important aspect of genomics is genotypic phasing, which has important functional implications. Although there are recently developed methods for whole genome haplotyping, it is still of interest to consider targeted haplotyping approaches in some situations. Methodologies such as allele-specific polymerase chain reaction and single molecule dilution have been proposed for this purpose more than 20 years ago. However, each of these methodologies presents particular difficulties or limitations. In this study, an alternative method for allele-specific amplification for targeted haplotyping is proposed.

Hypothesis

The method was presented in a hypothetical situation in the clinical setting, and can be divided into three steps: (1) Genotype all the markers (or sequence an entire target region); (2) If phase ambiguity is observed, amplify the entire region in a reaction combining two key factors: a non-extendable oligonucleotide probe that binds only to one of the alleles of one of the heterozygous markers, and a 5′→3′ exonuclease activity-lacking Taq DNA polymerase. By doing so, the reaction occurs having only one of the alleles as the template for amplification;(3) Genotype the other heterozygous markers to resolve phase ambiguity.

Discussion

The presented method, although a hypothesis (has not been formally tested for targeted haplotyping yet), is based on well-established concepts of polymerase chain reaction and probe-based single nucleotide polymorphism genotyping. It has the potential to overcome both allele-specific polymerase chain reaction (it has a highly discriminatory capacity, does not require labourious and time-consuming standardisation and can make use of genotyping assays validated for a huge number of single nucleotide polymorphisms in the genome) and single molecule dilution (it is technically more straightforward, less time-consuming and requires a simpler infrastructure), indicating its adequacy for haplotyping applications in the clinical setting.

Conclusion

Although there are other methods already proposed for allele-specific polymerase chain reaction for targeted haplotyping applications, the proposed probe-based method (which can be combined with sequencing for detecting or de novo variants) seems to be well capable of outperforming them as a straightforward, rapid, cheap and reliable assay, especially for applications in the clinical setting.

Introduction

The rapid advances in the knowledge of cancer (and other complex diseases) aetiology strongly suggests that cancer is (in non-familial cases) a multifactorial trait, being modulated by a rather complex interplay between germ-line genetic factors and genome-environment interactions[1]. Regarding genetic factors, most technologies and published studies do not take into account the genotypic phasing (i.e. the haplotypes underlying the observed genotypes; Figure 1) as commented elsewhere[2], which have been evidenced as of great functional relevance[3]. Among the earliest strategy in this regard is allele-specific polymerase chain reaction (AS-PCR), which consists of at least one of the primers annealing site overlapping-near to its 3' end-the variation location, resulting in primer binding or non-binding according to the allele under stringent amplification conditions[4]. Another early strategy was dilution of genomic DNA to single molecule level, in order to separate alleles physically[5]. These two methods are the basis for most of more recently proposed haplotyping techniques, although other approaches (such as single sperm cell sequencing) have also been proposed[6]. In fact, single molecule dilution has been used in recent studies that developed strategies for whole genome haplotyping at high quality standards, which are promising techniques to revolute cancer and human genomics at research and clinical levels[2,7,8].

An illustration of the phase ambiguity phenomenon in the presence of two heterozygous SNPs in the same target genomic region, resulting in two possible diplotypes coherent with the observed genotypes.

Although such whole-genome haplotyping methods are highly informative and described as of relatively low cost, they have some important limitations. First, the technique is relatively time-consuming, technically not straightforward and requires non-trivial infrastructure, thus limitingits applications especially in the clinical setting.In this regard, it is also of note that a genetic test in the clinical setting could be focused on specific regions rather than on the whole genome. In clinical research, a common interest is to scan a candidate region for a disease susceptibility locus (DSL) in unrelated individuals using association mapping, which relies on genotyping several markers [normally single nucleotide polymorphisms (SNPs)]in a defined genomic location. Even in a situation where interest lies in identifying a genetic profile that might be causing a new Mendelian syndrome observed in a group of related individuals with no a priori hypothesised candidate gene/locus, a whole-genome linkage mapping approach could be undertaken and, then, the identified genomic location would be further scanned for fine mapping the DSL by association mapping[9]. In genetic epidemiology research there could also be more interest in targeted-over whole-genome haplotyping in some situations, such as scanning a large region where genome-wide association is found for the causative DSL. Epidemiologically, there is the alternative of haplotype estimation by computational algorithms, although this approach also has important limitations[10]. To name a few, when sample size is not large, haplotype estimation by computational algorithms will be limited regarding the posterior probability (i.e. the uncertainty about the true diplotype at the individual level) and, consequently, the power to detect an association will be reduced (type II error). Moreover, it is necessary to estimate the haplotypes in stratified ethnic groups (which does not have an accurate definition or is not even meaningful for admixture populations) and disease status, since not doing so can lead to biased estimates, thus reducing the sample size for haplotype estimation.

The aforementioned information indicates that there is a need for targeted haplotyping, especially where interest lies in specific genomic regions. Although techniques based on AS-PCR has been proposed for targeted haplotyping, its rationale is known to be subjected to several difficulties that impose limitations such as a laborious and time-consuming standardisation, a complicated balance between amplification sensitivity and specificityand, more crucially, difficulties for efficient allele discrimination[11]. Such difficulty was acknowledged by and can be seen in publications proposing the use of AS-PCR for phase determination of SNPs[12,13]. These limitations do not apply to single molecule dilution methods. However, these are technically less straightforward methods (requiring specifically trained human resources) that require more infrastructure and are more time-consuming[14]. Given the exposed above, there is a clear need for accurate, technically simple, rapid and economically cheaper haplotyping methodology for situations where interest lies in specific regions (i.e. targeted haplotyping) for both research and clinical applications. Here, an alternative method for AS amplificationfor targeted haplotyping applications is proposed.

Hypothesis

A three-step probe-based method for targeted haplotyping

The methodology is straightforward and based on very well-established PCR and genotyping concepts. Basically, genotypic information (obtained from commonly used genotyping assays) is used to generate several copies of the targeted region by a regular PCR, having a single allele as the template. The ASamplicons can then be re-genotyped, allowing for direct haplotyping. To better illustrate the methodology, the following hypothetical situation in the clinical setting is used: a patient has a familial history of a cancer type that is known to be highly associated with a specific haplotype composed of specific alleles of four SNPs (SNP1, SNP2, SNP3 and SNP4) in a given genomic region. In such a case, it would be advisable to apply a genetic test for the presence of the disease-associated haplotype. According to the proposed haplotyping methodology, the patient’s genomic DNA will be submitted to the following procedures. The first step is to genotype all four SNPs and verify whether or not there is phase ambiguity (i.e. two or more are SNPs in heterozygosis, as illustrated in Figure 1). If there is not, the haplotypes can be inferred without uncertainty (excluding, of course, technical errors). If there is phase ambiguity, it is necessary to determine which SNP alleles are in the same chromosome, which is not possible from the DNA sample directly, since it can be understood as a (expectedly) 1:1 mixture of the two alleles (Figure 1). In the hypothetical example, of the four SNPs, two were heterozygous (the genotypes were Aa. BB, cc and Dd for SNP1, SNP2, SNP3 and SNP4, respectively)resulting in phase ambiguity.

To overcome this situation, the next (second) step is to amplify the whole region (i.e. generate an amplicon that contains the four SNPs) in a PCR reaction with two key factors: a non-extendable oligonucleotide probe that binds to only one of the alleles of one of the heterozygous SNPs and a 5’→3’ exonuclease activity-lacking Taq DNA polymerase (Figure 2). In the hypothetical example, a probe that binds to the ‘A’ but not to the ‘a’ allele of SNP1 was used in a PCR reaction with a 5’→3’ exonuclease activity-lacking Taq DNA polymerase. This resulted in blocking the extension of the DNA strands being synthesised from the chromosome that contains the ‘A’ allele, while the selected region can be normally amplified from the other chromosome (i.e. the one that contains the ‘a’ allele). The implication is the amplification of the target region having a single chromosomal allele as the template. In the final step, this PCR product can be used in a nested reaction(s) to re-genotype the other heterozygous markers. In the example, a reaction using the product from the previous step as the template was used to re-genotype SNP4, yielding a ‘homozygous dd’ genotype. Such homozygosis, in fact, indicates which allele of SNP4 was co-inherited with the ‘a’ allele. There is now enough information to unambiguously determine the patient’s diplotype, which is ABcD/aBcd.

An illustrative representationofthe proposed methodology for allele-specific amplification for targeted haplotyping. In the first step, all variants are genotyped(represented by amplifying and sequencing the whole target region, although any other genotyping method, such as probe-based genotyping, would be applicable). If phase ambiguity is observed, a PCR to amplify the target region (step 2) is performed using a non-extendable oligonucleotide probe and a 5’→3’ exonuclease activity-lacking Taq DNA polymerase, allowing a highly-specific amplification of only one of the alleles.

Evaluation of hypothesis

Considering that the manuscript proposes a technique, evaluating it would consist of performing the technique. An important consideration in this regard is the standard technique to compare the results of the proposed method, since there are a variety of proposed approaches for haplotyping. One approach that could be used is sequencing of single-sperm cells[6], asthis method ensures that only one chromosome will be sequenced. To overcome the limitation of recombination occurring within the haplotype, a short region known to present very high linkage disequilibrium could be used. By comparing the haplotyping results for different male individuals using single-sperm cells sequencing and the proposed method (using DNA sources such as peripheral blood), the last would be properly evaluated regarding its concordance (estimated using metric such as the kappa coefficient) with the standard.

Consequences of hypothesis

Assuming the proposed method is efficient, it would consist of a useful tool for haplotyping in both research and clinical settings. Regarding research, the main applicability would be in genetic epidemiology studies, especially in relatively small samples (in which phase inference is not precise). This would be particularly important for diseases such as some types of rare cancers, which are unlikely to be studied in samples where the number of cases is large. Moreover, considering that probe-based methods are easily scalable into platforms for fast processing, the proposed method could also be used to focus on a given genomic locus that presented a number of hits in a genome-wide association study. In the clinical setting, this method would be applicable in the context of personalized medicine, allowing simple, fast and economically cheap (as discussed below) targeted-haplotyping of genomic regions known to be highly associated with a given disease or any health outcome in general.

Discussion

It is important to note that the concept of AS amplification presented here is not innovative, since AS-PCR has been conceived several years ago. The innovation of the proposed methodology is to take advantage of the preferable properties of probe-based over primer mismatch-based allelic detection. The use of a probe allows the use of a single primer pair (thus eliminating the laborious process of finding a suitable balance between amplification specificity and sensibility), and there are validated probes for SNP genotyping (i.e., allele-detection) at several loci in the genome. As commented in the first section, there are other techniques proposed for targeted haplotyping, such as diluting the DNA sample to approximately a single copy with final detection by techniques as mass spectrometry[14]. Although this technique has the advantage of not requiring previous amplification of the genomic region of interest (allowing haplotypingof very large regions), it consists of a relatively labourious and complicated method (requiring more infrastructure and training) when compared to straightforward and cheap PCR-based steps. Moreover, there are 5’→3’ exonuclease activity-lacking Taq DNA polymerases capable of generating fragments ≥ 20 kb commercially available, and joining two or more PCR products by adding restriction sitesatthe 5’ ends of primers is also straightforward and allows the generation of large AS genomic regions.

The importance of the proposed probe-based method is on its practical simplicity and adequacy for the clinical setting and epidemiological research. In practice, the method is based on a series of a few PCR reactions, requiring limited infrastructure and training. In fact, PCR, in addition of being one of the most popular techniques in molecular biology,is a tool already present in the clinical setting for genetic testing. It outperforms primer mismatch-based methods (i.e. classical AS-PCR) for being a more specific and reliable technique that does not require extensive (and sometimes unsuccessful) standardisation, and outperforms dilution-based approaches for being simpler (both in theory and in practice), faster, cheaper and also capable of analysing large genomic regions.

Conclusion

The proposed method for targeted haplotyping has notable simplicity. The basis of this method is to explore the availability of 5’→3’ exonuclease activity-lacking Taq DNA polymerases to make the amplification reaction sensible to the presence of a probe, allowingtheamplification of a specific allele. Although it is yet to be tested in practice,the methodology is based on very well-established concepts, mainly the use of an AS probe and the requirement of 5’→3’ exonuclease activity for the Taq DNA polymerase to remove a non-extendable oligonucleotide probe (both concepts form the basis of probe-based genotyping assays). Although there are other methods already proposed for AS-PCR for targeted haplotyping applications, the proposed probe-based method (which can be combined with sequencingfor detecting or de novo variants) seems to be well capable of outperforming themas a straightforward,rapid, cheap and reliable assay, especially for applications in the clinical setting.

Author Contribution

All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.

Competing interests

None declared.

Conflict of interests

None declared.

A.M.E

All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

References

  • 1. Peltomaki P . Mutations and epimutations in the origin of cancer. Exp Cell Res 2012 Feb;318(4):299-310.
  • 2. Peters BA, Kermani BG, Sparks AB, Alferov O, Hong P, Alexeev A. Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells. Nature 2012 Jul;487(7406):190-5.
  • 3. Tewhey R, Bansal V, Torkamani A, Topol EJ, Schork NJ. The importance of phase information for human genomics. Nat Rev Genet 2011 Mar;12(3):215-23.
  • 4. Ruano G, Kidd KK. Direct haplotyping of chromosomal segments from multiple heterozygotes via allele-specific PCR amplification. Nucleic Acids Res 1989 Oct;17(20):8392.
  • 5. Ruano G, Kidd KK, Stephens JC. Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. ProcNatlAcadSci U S A 1990 Aug;87(16):6296-300.
  • 6. Kirkness EF, Grindberg RV, Yee-Greenbaum J, Marshall CR, Scherer SW, Lasken RS. Sequencing of isolated sperm cells for direct haplotyping of a human genome. Genome Res 2013 May;23(5):826-32.
  • 7. Fan HC, Wang J, Potanina A, Quake SR. Whole-genome molecular haplotyping of single cells. Nat Biotechnol 2011 Jan;29(1):51-7.
  • 8. Kaper F, Swamy S, Klotzle B, Munchel S, Cottrell J, Bibikova M. Whole-genomehaplotyping by dilution, amplification, and sequencing. ProcNatlAcadSci U S A 2013 Apr;110(14):5552-7.
  • 9. Laird M, Lange C. The fundamentals of modern statistical genetics. New York: Springer 201167-86.
  • 10. Salem RM, Wessel J, Schork NJ. A comprehensive literature review of haplotyping software and methods for use with unrelated individuals. Hum Genomics 2005 Mar;2(1):39-66.
  • 11. Ayyadevara S, Thaden JJ, Shmookler Reis RJ. Discrimination of primer 3’-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction. Anal Biochem 2000 Aug;284(1):11-8.
  • 12. Pettersson M, Bylund M, Alderborn A. Molecular haplotype determination using allele-specific PCR and pyrosequencing technology. Genomics 2003 Sep;82(3):390-6.
  • 13. Yu CE, Devlin B, Galloway N, Loomis E, Schellenberg GD. ADLAPH: a molecular haplotyping method based on allele-discriminating long-range PCR. Genomics 2004 Sep;84(3):600-12.
  • 14. Ding C, Cantor CR. Direct molecular haplotyping of long-range genomic DNA with M1-PCR. ProcNatlAcadSciU S A 2003 Jun;100(13):7449-53.
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Keywords