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SNP 分型技术之一:High Resolution Melting (HRM) analysis

已有 15598 次阅读 2010-12-29 09:51 |个人分类:未分类|系统分类:科研笔记

高分辨率熔解(High-resolution melting,简称HRM)分析是一门新兴的技术。它仅仅通过PCR之后的熔解曲线分析,就能检测PCR片段的微小序列差异,从而应用在突变扫描、序列配对和基因分型等多个方面。凭借其速度和简便性,高分辨率熔解这种方法正在迅速普及。


其实双链DNA的熔解分析可追溯到上世纪六十年代,当时是通过紫外吸收来监测的。分析需要几微克的DNA,而样品以0.1-1.0°C/分钟的速率缓慢加热,常常要花上几个小时才能完成。后来,LightCycler定量PCR仪的出现,让荧光熔解分析变得流行。样品量因毛细管而缩减至纳克级,且熔解速率更快,达0.1-1.0°C/秒,这样熔解时间缩短至几分钟。当时使用的染料是SYBR® Green I。


然而,这种熔解分析只能区分在片段大小和GC含量上差别较显著的DNA序列,比如检查PCR扩增产物中是否存在引物二聚体及其他非特异性的扩增。如果要区分SNP,分辨率还不够。为什么呢?这里有必要先介绍一下饱和染料和非饱和染料。SYBR Green I就属于非饱和性染料,由于它对PCR的抑制作用,在实验中的使用浓度很低,远未将DNA双螺旋结构中的小沟饱和。这样,DNA双链高温变性时,单链部分的荧光染料分子发生重排,荧光染料分子重新结合到双链DNA的空置位点,造成荧光信号没有变化,因此出现假阴性,特异性下降。


于是,饱和染料问世了。为什么称为饱和染料呢?因为它们即便在饱和浓度(荧光最大)下,也不会抑制PCR。故高浓度的染料饱和了DNA双螺旋结构中的小沟,在DNA解链过程中就不会发生重排,这样熔解曲线才有了更高的分辨率。目前市场上的饱和染料主要有LC Green、LC Green Plus、SYTO 9等几种。光有染料还不够,仪器也要相应升级呢。


扩增子的熔解曲线完全取决于DNA碱基序列。序列中如有一个碱基发生了突变,都会改变DNA链的解链温度。但是这个差异极小,只有零点几摄氏度,如果仪器的分辨率不高,是根本无法检测的。
那么什么样的分辨率才是高分辨率呢?根据仪器现有的功能测试,在熔解操作时每摄氏度至少要获得10个数据点,这是对仪器的最低要求。此外,温度均一性也同样重要。如果两孔之间温度相差0.1°C,就很可能导致最终的熔解温度相差0.1°C,这样就无法保证HRM分析结果的准确性。大多数常规定量PCR仪的孔间温度差在0.3-0.5°C,这也决定了它们无法胜任HRM。


目前市场上有多个厂家提供HRM分析的仪器,包括Idaho Technology、Corbett(QIAGEN)、Roche等,有些是专供HRM的仪器,有些则是附带HRM功能的定量PCR仪。HRM的发明者—美国犹他大学医学院的Wittwer实验室曾评估了市场上多台仪器在基因分型和突变扫描上的性能1。他们发现,对于大部分仪器的准确基因分型来说,主要限制在于平板上的空间温度差异(Tm的标准偏差为0.020-0.264°C)。其它差异如数据密度、信噪比和熔解速率,也会影响杂合体的扫描。经过比较发现,空气加热型仪器以及样品温度单独控制的仪器有着更低的Tm标准偏差(0.018-0.065),而加热块型仪器则在0.092-0.274。


在拥有饱和染料和高分辨率仪器之后,HRM分析就可以开始啦。这个过程其实很简单,就是对PCR的扩增子进行加热,温度从50°C逐渐上升到95°C。在此过程中,扩增子逐渐解链,在到达熔解温度(Tm)时,DNA链完全分开。在HRM分析的初期,荧光强度很高,随着温度升高,双链DNA逐渐减少,荧光强度也就下降了。HRM仪器通过照相机,记录下荧光变化的整个过程。通过对数据的作图,就生成了熔解曲线。


果然够简单吧,连探针也无需设计,只要在常规PCR中加一个饱和染料即可。HRM既可以对未知突变进行筛查、扫描,也可以对已知突变进行分析。相对于传统的突变分析而言,操作步骤大大简化,时间和成本也降低了不少。而且样品经PCR扩增后直接进行HRM分析,无需转移,真正实现了闭管操作,降低了污染的风险。


正是凭借这些优势,HRM近年来广泛用于突变扫描、序列配对、基因分型和甲基化分析等应用中。


序列配对


序列差异分析常用的方法包括单链构象多态性、变性梯度凝胶电泳、变性高效液相色谱、测序等,这些方法都需要分离样品,之后进行多步反应,操作繁琐,耗时长,且PCR产物有污染的风险。而HMR在5分钟之内就能完成,无需额外的步骤,也容易开发成高通量筛选,且是唯一的闭管操作,增加了分析的可靠性,这些优势对于分子诊断分析而言,格外重要。


在器官移植中,医生通常会检测兄弟姐妹的HLA,以便了解主要组织相容性是否匹配。利用PCR产物的熔解曲线,能快速配对HLA序列。与熔解温度(Tm)不同,那只是熔解曲线上的一个点,高分辨率熔解是分析整个熔解曲线。利用熔解曲线的形状作为指示剂,来显示杂合DNA所形成的异源双链的序列匹配。之后将两个DNA按1:1的比例混合,重新熔解,并将新曲线与原先的曲线进行比较,来验证序列的匹配。如果两个样品的序列不是完全相同的,那么混合后异源双链的熔解曲线形状会改变。


瑞典乌普萨拉大学的Olof Gidlöf等曾开发出线粒体基因组中两个超变区HVI和HVII的HRM分析,以分辨不同个体的DNA,作为法医鉴定中测序前的预筛选2。研究结果表明,线粒体DNA中HVI和HVII所存在的序列变异确实产生了熔解曲线形状的差异,能辨别不同个体的DNA,这样就能排除不匹配的法医材料,从而节省线粒体DNA测序的时间和费用。另外,与其它技术相比,HRM操作简单,价格低,能同时筛选大量的样本。


突变扫描


单碱基改变、插入、缺失都能通过HRM来检测。在灵敏度和特异性方面,高分辨率熔解也高于dHPLC在内的传统方法。当变异体为低等位基因片段时,它甚至比DNA测序还灵敏。测序只能检测低至20%的变异体,而HRM能检测的变异体低至2%。而且,测序所花的时间、费用和精力,与HRM是不可同日而语的。


对于400 bp以下的PCR产物,扫描单碱基变化的灵敏度和特异性皆为100%。对于400到1000 bp的PCR产物,灵敏度为96.1%,特异性为99.4%。PCR产物中变异的位置不影响扫描准确性。尽管HRM是为检测杂合体(一个等位基因发生突变)而设计的,但它也能检测纯合体(两个等位基因都有突变)。对于半合子变异体(X连锁或Y染色体),最好将未知样品与已知野生型的样品混合。


LMNA基因的突变会导致多种失调,现在称之为核纤层蛋白综合征。由于待研究的个体颇多,故必须用一种特别灵敏且特异的方法来进行突变扫描。于是,Gilles Millat等就开发出适合LMNA突变扫描的HRM分析3。他们同时用了HRM和dHPLC两种方法,对64位患有扩张性心肌病的病人进行筛选。结果表明,凡是dHPLC或直接测序能检测出的基因变异,HRM分析也能轻松鉴定,且速度比dHPLC快2倍,还更为经济。研究人员认为,HRM分析是鉴定LMNA遗传变异的高灵敏、高通量的廉价方法。


基因分型


传统的基因分型方法不仅耗时费力,还需要购买价格不菲的探针。相比之下,HRM分析则是优势多多,无需扩增和制备,排除了污染的风险;探针的钱也省了;还能检测未知的变异体,这一点探针可无能为力。有了饱和染料,PCR产物全程被标记,因此所有的熔解区域都能检测到。对于同一扩增子内的不同杂合体,通过曲线形状的差异也能区分开。HRM大约能分辨93%的杂合体。大部分纯合体也能通过熔解来分辨,但不是全部。大约有4%的人单碱基变异体有着相同的Tm,且图像对称。在这种情况下,可以将已知的纯合体与未知的纯合体混合,再进行分析。


Lasse Kristensen等曾为参与甲基代谢的关键基因(MTHFR、MTR和DNMT3b)开发出HRM分析,对它们进行基因分型,并在Rotor-gene 6000等仪器上进行检测4。他们认为,与基于TaqMan探针的基因分型相比,HRM分析更经济。无需使用探针,分析所需的优化也更少,成功率更高。与焦磷酸测序、SNP芯片等多种技术相比,HRM是最佳选择。


甲基化分析


HRM分析还为DNA甲基化状态的检测另辟蹊径。DNA样品通过亚硫酸氢盐的处理,将未甲基化的胞嘧啶转化成尿嘧啶。这样,原先未甲基化模板所产生的PCR产物比甲基化模板的Tm要低。HRM还能确定特定样品中甲基化的比例。将不同比例的甲基化和未甲基化DNA混合,制作出标准曲线,将样品的熔解曲线与之相比较,从而确定样品中甲基化的比例。


异常的甲基化模式往往与癌症相关联,于是Erci Smith等开发并验证了快速定量DNA甲基化状态的HRM分析5。他们利用数学方法来标准化加热DNA所产生的原始荧光数据,从这些数据中计算出熔解开始和结束的温度,从而反映模板分子的甲基化水平。之后,他们还用已知甲基化水平的寡核苷酸及DNA进行了验证。他们发现,通过HRM分析这种快速单管的方法来定量甲基化是可靠的,引物容易设计,且无需专门的软件。所获得的参数提供了标本中甲基化的客观描述和定量,能用于标本之间的统计学比较。


综上,HRM技术应用广泛,操作简单又经济,结果精确且可靠,适合任何实验室使用。市场上有一些专供HRM分析的仪器,不过,那些带有HRM功能的定量PCR仪则更实用。QIAGEN的Rotor-Gene Q(Corbett Rotor-Gene 6000)就是全球第一台将实时扩增与HRM分析技术相结合的实时荧光定量分析装置。Rotor-Gene Q的温度均一性为0.01°C,温度分辨率为0.02°C,解决了温度均一性、精确性和分辨率的问题,实现了定量扩增与HRM的合二为一。


 

High Resolution Melting (HRM) analysis
 
In molecular biology High Resolution Melt or HRM analysis, as it will be referred to herein, is a powerful technique for the detection of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah[1]. It has advantages over other genotyping technologies. Namely:
 
It is cost effective vs. other genotyping technologies such as sequencing and Taqman SNP typing. This makes it ideal for large scale genotyping projects.
It is fast and powerful thus able to accurately genotype many samples rapidly.
It is simple. With a good quality HRM assay, powerful genotyping can be performed by non-geneticists in any laboratory with access to an HRM capable real-time PCR machine.
[edit] How does high resolution melting analysis work?
HRM analysis is performed on double stranded DNA samples. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. Essentially the PCR process turns a tiny amount of your region of DNA of interest in to a large amount, so you have quantities large enough for better analysis. In the tube there are now many of copies of your region of DNA of interest. This region that is amplified is known as the amplicon. After the PCR process the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50?C up to around 95?C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or “melt” apart.
 
 
 
The secret of HRM is to monitor this process happening in real-time. This is achieved by using a fluorescent dye. The dyes that are used for HRM are known as intercalating dyes and have a unique property. They bind specifically to double-stranded DNA and when they are bound they fluoresce brightly. In the absence of double stranded DNA they have nothing to bind to and they only fluoresce at a low level. At the beginning of the HRM analysis there is a high level of fluorescence in the sample because of the billions of copies of the amplicon. But as the sample is heated up and the two strands of the DNA melt apart, presence of double stranded DNA decreases and thus fluorescence is reduced. The HRM machine has a camera that watches this process by measuring the fluorescence. The machine then simply plots this data as a graph known as a melt curve, showing the level of fluorescence vs the temperature:
 
 
 
Spot the difference The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. It is dependent on the sequence of the DNA bases. If you are comparing two samples from two different people, they should give exactly the same shaped melt curve. However if one of the people has a mutation in the DNA region you have amplified, then this will alter the temperature at which the DNA strands melt apart. So now the two melt curves appear different. The difference may only be tiny, perhaps a fraction of a degree, but because the HRM machine has the ability to monitor this process in “high resolution”, it is possible to accurately document these changes and therefore identify if a mutation is present or not.
 
 
 
Wild type, heterozygote or homozygote? Things become slightly more complicated than this because organisms contain two (or more) copies of each gene, known as the two alleles. So, if a sample is taken from a patient and amplified using PCR both copies of the region of DNA (alleles) of interest are amplified. So if we are looking for mutation there are now three possibilities:
 
Neither allele contains a mutation
One or other allele contains a mutation
Both alleles contain a mutation.
These three scenarios are known as “Wild –type”, “Heterozygote” or “Homozygote” respectively. Each gives a melt curve that is slightly different. With a high quality HRM assay it is possible to distinguish between all three of these scenarios.
 
 
 
[edit] Applications of HRM
SNP typing/Point mutation detection
Conventional SNP typing methods are typically time consuming and expensive, requiring several probe bases assays to be multiplexed together or the use of DNA microarrays. HRM is more cost effective and reduces the need to design multiple pairs of primers and the need to purchase expensive probes. The HRM method has been successfully used to detect a single G to A substitution in the gene Vssc (Voltage Sensitive Sodium Channel) which confers resistance to the acaricide permethrin in Scabies mite. This mutation results in a coding change in the protein (G1535D). The analysis of scabies mites collected from suspected permethrin susceptible and tolerant populations by HRM showed distinct melting profiles. The amplicons from the sensitive mites were observed to have a higher melting temperature relative to the tolerant mites, as expected from the higher thermostability of the GC base pair [2]
In a field more relevant to clinical diagnostics, HRM has been shown to be suitable in principle for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. More than 400 mutations have been identified in these genes.
The sequencing of genes is the gold standard for identifying mutations. Sequencing is time consuming and labour intensive and is often preceded by techniques used to identify heteroduplex DNA, which then further amplify these issues. HRM offers a faster and more convenient closed-tube method of assessing the presence of mutations and gives a result which can be further investigated if it is of interest. In a study carried out by Scott et al. in 2006 [3], 3 cell lines harbouring different BRCA mutations were used to assess the HRM methodology. It was found that the melting profiles of the resulting PCR products could be used to distinguish the presence or absence of a mutation in the amplicon. Similarly in 2007 Krypuy et al. [4]. showed that the careful design of HRM assays (with regards to primer placement) could be successfully employed to detect mutations in the TP53 gene, which encodes the tumour suppressor protein p53 in clinical samples of breast and ovarian cancer. Both these studies highlighted that fact that changes in the melting profile can be in the form of a shift in the melting temperature or an obvious difference in the shape of the melt curve. Both of these parameters are a function of the amplicon sequence. The overall consensus is that HRM is a cost efficient method that can be employed as an initial screen for samples suspected of harbouring polymorphisms or mutations. This would reduce the number of samples which need to be investigated further using more conventional methods.
 
Zygosity testing
Currently there are many methods used to determine the zygosity status of a gene at a particular locus. These methods include the use of PCR with specifically designed probes to detect the variants of the genes (SNP typing is the simplest case). In cases where longer stretches of variation is implicated post PCR analysis of the amplicons may be required. Changes in enzyme restriction, electrophoretic and chromatographic profiles can be measured. These methods are usually more time consuming and increase the risk of amplicon contamination in the laboratory, due to the need to work with high concentrations of amplicons in the lab post-PCR. The use of HRM reduces the time required for analysis and the risk of contamination. HRM is a more cost effective solution and the high resolution element not only allows the determination of homo and heterozygosity, it also resolves information about the type of homo and heterozygosity, with different gene variants giving rise to differing melt curve shapes. A study by Gundry et al. 2003 [5], showed that fluorescent labelling of one primer (in the pair) has been shown to be favourable over using an intercalating dye such as SYBR green I. However, progress has been made in the development and use of improved intercalating dyes [6] which reduce the issue of PCR inhibition and concerns over non-saturating intercalation of the dye.
 
Epigenetics
The HRM methodology has also been exploited to provide a reliable analysis of the methylation status of DNA. This is of significance since changes to the methylation status of tumour suppressor genes, genes that regulate apoptosis and DNA repair, are characteristics of cancers and also have implications for responses to chemotherapy. For example, cancer patients can be more sensitive to treatment with DNA alkylating agents if the promoter of the DNA repair gene MGMT of the patient is methylated. In a study which tested the methylation status of the MGMT promoter on 19 colorectal samples, 8 samples were found to be methylated [7]. Methylated DNA can be treated by bi-sulphite modification, which converts non-methylated cytosines to uracil. Therefore, PCR products resulting from a template that was originally unmethylated will have a lower melting point than those derived from a methylated template. HRM also offers the possibility of determining the proportion of methylation in a given sample, by comparing it to a standard curve which is generated by mixing different ratios of methylated and non-methylated DNA together. This can offer information regarding the degree of methylation that a tumour may have and thus give an indication of the character of the tumour and how far it deviates from what is “normal”. HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted to high throughput screening testing, and again it minimises the possibility of amplicon spread and contamination within a laboratory, owing to its closed-tube format.
 
Intercalating dyes
To follow the transition of dsDNA (double-stranded) to ssDNA (single-stranded), intercalating dyes are employed. These dyes show differential fluorescence emission dependent on their association with double-stranded or single-stranded DNA. SYBR Green I is a first generation dye for HRM. It fluoresces when intercalated into dsDNA and not ssDNA. Because it may inhibit PCR at high concentrations, it is used at sub-saturating concentrations. Recently, some researchers have discouraged the use of SYBR Green I for HRM [8], claiming that substantial protocol modifications are required. This is because it is suggested that the lack of accuracy may result from “dye jumping”, where dye from a melted duplex may get reincorporated into regions of dsDNA which had not yet melted [5][8]. New saturating dyes such as LC Green and LC Green Plus , ResoLight, EvaGreen, Chromofy and SYTO 9 are available on the market and have been used successfully for HRM. However, some groups have successfully used SYBR Green I for HRM with the Corbett Rotorgene instruments [9] and advocate the use of SYBR Green I for HRM applications.
 
[edit] References
^ for academic treatment of the history of HRM see http://www.dna.utah.edu/Hi-Res/TOP_Hi-Res%20Melting.html
^ Pasay C, Arlian L, Morgan M, et al. (March 2008). "High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites". Med. Vet. Entomol. 22 (1): 82–8. doi:10.1111/j.1365-2915.2008.00716.x. PMID 18380658. http://www3.interscience.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0269-283X&date=2008&volume=22&issue=1&spage=82. 
^ James PA, Doherty R, Harris M, et al. (February 2006). "Optimal selection of individuals for BRCA mutation testing: a comparison of available methods". J. Clin. Oncol. 24 (4): 707–15. doi:10.1200/JCO.2005.01.9737. PMID 16446345. http://jco.ascopubs.org/cgi/content/full/24/4/707. 
^ Krypuy M, Ahmed AA, Etemadmoghadam D, et al. (2007). "High resolution melting for mutation scanning of TP53 exons 5-8". BMC Cancer 7: 168. doi:10.1186/1471-2407-7-168. PMID 17764544. PMC 2025602. http://www.biomedcentral.com/1471-2407/7/168. 
^ a b Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT (March 2003). "Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes". Clin. Chem. 49 (3): 396–406. PMID 12600951. http://www.clinchem.org/cgi/pmidlookup?view=long&pmid=12600951. 
^ Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (June 2003). "High-resolution genotyping by amplicon melting analysis using LCGreen". Clin. Chem. 49 (6 Pt 1): 853–60. PMID 12765979. http://www.clinchem.org/cgi/content/full/49/6/853. 
^ Wojdacz TK, Dobrovic A (2007). "Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation". Nucleic Acids Res. 35 (6): e41. doi:10.1093/nar/gkm013. PMID 17289753. PMC 1874596. http://nar.oxfordjournals.org/cgi/content/full/35/6/e41. 
^ a b Reed GH, Kent JO, Wittwer CT (June 2007). "High-resolution DNA melting analysis for simple and efficient molecular diagnostics". Pharmacogenomics 8 (6): 597–608. doi:10.2217/14622416.8.6.597. PMID 17559349. http://www.futuremedicine.com/doi/abs/10.2217/14622416.8.6.597?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dncbi.nlm.nih.gov. as PDF
^ Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T (June 2008). "Detection of alpha-thalassemia-1 Southeast Asian type using real-time gap-PCR with SYBR Green1 and high resolution melting analysis". Eur. J. Haematol. 80 (6): 510–4. doi:10.1111/j.1600-0609.2008.01055.x. PMID 18284625. http://www3.interscience.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0902-4441&date=2008&volume=80&issue=6&spage=510.




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