基于LDR对高度降解和微量DNA检材的SNPs分型研究
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摘要
背景与目的
最近二十年来法医物证学最引人注目的变革,就是DNA分析技术的应用及发展。对于高度降解检材和微量检材,如何从中获取可靠的、足量的遗传学信息,来进行法医学个体识别和亲权鉴定,一直是法医物证学面临的重大挑战。
随着降解DNA分析技术的不断发展,如何评价、比较不同方法的检验效能的问题,逐渐引起法医学者的关注;要实施此类研究,首先需要制备出特定片段长度的降解DNA标准样品。迄今,已提出了几种降解DNA的人工制备方法,包括超声振荡、DNA酶消化以及其它理化措施(改变环境温度、湿度、自然放置等),但是,这些方法目前均未被广泛接受。因此,本文尝试探讨高度降解检材的人工制备方法。
随着相关研究的不断深入,尽可能缩短靶DNA片段的扩增子长度,已经成为降解DNA分析的基本策略,目前mini-STR-PCR和mini-sequencing是其代表技术。但是,现有的mini-STR-PCR和mini-sequencing技术要求降解DNA片段的长度不得小于100bp～200bp。一旦基因组DNA发生了非常严重的降解,片段小于100bp的情况下,如何获得足量、可靠的遗传学信息来进行法医学鉴定呢?对此问题,目前尚无确切的解决方法。
当前,对于微量检材或低拷贝数检材(low copy number,LCN)的DNA分析,主要有两种检测策略:一是增强PCR-STR分型法,主要是增加PCR反应循环次数,提高STR位点扩增成功率(检测信号多寡)和产物量(检测信号的强弱),而后进行DNA分析;二是首先进行LCN检材的全基因组扩增或者巢式PCR扩增,以获取足量的DNA模板,而后进行DNA分型。但是,由于LCN检材所含DNA模板量过少,受随机效应的影响,采用上述两种策略,均会出现“额外”等位基因、影子带(stutter带)、等位基因丢失等一些难以避免的问题,STR分型结果通常具有不可重复性,并难以评价其有效性,无法得到可靠的法医学鉴定结论。
近年来,基于连接酶检测反应(LDR)的SNPs分型技术快速发展,为降解检材和微量检材的法医学DNA分析提供了新的契机。首先,LDR探针与模板DNA互补结合的序列不需过长,只要满足以下两个条件即可:(1)能够有效识别待测靶位点,(2)不影响连接酶的识别与结合所需片段长度,所以,LDR探针长度不超过数十个碱基。再者,对于双链DNA的单链切口,DNA连接酶的识别与连接反应具有高度严谨性。因此,从理论上说,只要DNA片段不小于30～50bp,即可采用LDR技术进行SNP分型检测,这一特性尤其适用于高度降解DNA分析。所以,本文尝试LDR-PCR策略来检测高度降解DNA。
再者,锁式探针(padlock probes)可识别靶位点并形成包含单链切口的局部双螺旋结构,经LDR反应可生成环状模板,LDR反应的高度严谨性保证了等位基因识别的准确性;同时,超分支滚环扩增反应具有极强的扩增效能,可极大地放大LDR检测信号。将二者结合起来,对LCN检材的分析效能将得以极大提高。所以,本文拟开发LDR-HRCA策略来检测LCN检材。
材料与方法
枸橼酸钠抗凝人末梢静脉血样品12份,男女各6份,由中国医科大学法医学院法医血清学教研室提供。采用酚—氯仿法提取基因组DNA。
分别使用超声法和DNaseⅠ酶切法处理高分子量基因组DNA,于不同时间段取样,电泳和/或PCR扩增检测DNA片段长度,人工制备不超过100bp的高度降解DNA检材。
使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit对降解DNA检材进行复合STR分型检验。选择4个待测SNPs位点(rs17750303、rs2307647、rs2307557和rs17250992),并构建各位点的LDR探针:首先采用LDR反应,识别降解检材各待测位点的基因型;而后采用PCR反应,扩增连接后LDR探针,根据PCR产物电泳结果判断各位点基因型。
使用稀释法制备DNA浓度梯度样品:200、100、50、40、30、20、10pg/μl。使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit对微量检材进行复合STR分型检验。选择rs17750303位点,并构建一对等位基因特异性锁式探针(CPP和APP);首先采用LDR反应,识别待测位点的基因型;而后采用HRCA反应,扩增环化锁式探针,根据电泳结果判断各位点基因型。
结果
1、超声法处理基因组DNA,可重复性不佳,DNA片段分布范围表现出明显差异。随着DNaseⅠ酶切时间延长,DNA片段长度不断缩短;酶切150min,降解DNA片段长度不超过100bp。
2、对于片段长度不超过100bp的人工降解DNA样品,AmpFl STR(?)Identifiler(?)PCR Amplification Kit中全部STR基因座均不能识别。采用LDR-PCR方法,进行单位点检测,单碱基置换变异(rs17750303和rs17250992)和三碱基缺失/插入变异(rs2307647和rs2307557)均能得到正确分型结果,多态性碱基的变异类型不是LDR反应忠实性的主要影响因素;短LDR探针(rs17750303和rs2307647)与长探针(rs2307557和rs17250992)均可得到准确分型结果,探针特异性互补序列可短至10个核苷酸而不影响LDR反应。进行2位点及4位点复合检测,均可获得准确分型结果,可有效避免探针间交互效应。
3、使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit,PCR循环数34次,DNA模板量≥100ng,全部STR基因座均能正确识别;DNA模板量≤50ng,等位基因丢失现象严重,长扩增子座位尤为显著。采用LDR-HRCA方法检测rs17750303纯合型样品(CC型、AA型),产物量随DNA模板量降低而减少,20pg以上可获得正确的基因分型结果;CC纯合型样品10pg组共10份,CC表型7份,AC表型2份,无产物1份;AA纯合型样品10pg组共10份,AA表型6份,AC表型1份,CC表型1份,无产物2份。检测rs17750303杂合型样品(AC型),产物量随DNA模板量降低而减少,30 pg以上可获得正确的基因分型结果;10pg组共10份,AC表型6份,CC表型1份,AA表型2份,无产物1份。所有样品的检验过程中,均未发现影子带现象。
结论
1、采用DNaseⅠ酶切法处理基因组DNA,操作简单,DNA降解程度易于控制,结果稳定,可用于制备特定片段长度的降解DNA标准样品。
2、对于片段长度小于100bp的高度降解DNA检材,LDR-PCR方法可准确进行SNPs基因座分型,并可进行4个基因座复合分型检测。本方法对于高度降解检材的法医学DNA分析,具有较大的开发价值。
3、对于LCN检材,LDR-HRCA方法检测特异性好,灵敏度高于增强PCR-STR技术,可能更适用于法医学微量DNA分析。
4、本研究总结的待测SNPs位点筛选及相应LDR探针构建原则,可保证高度降解检材和微量检材SNPs分型的特异性和灵敏性,并且能够满足高通量检测的要求。
Background and objectives
For the past decades,the use of DNA analysis must be the most significant reformation in forensic fields.However,DNA degradation and trace DNA have always been special challenges for forensic scientists obtaining reliable and sufficient genetic information for personal identification and parentage testing.
To improve and assess the performance of different genotyping technologies in those cases where DNA is degraded,it would be desirable to have a reference sample of degraded DNA with known fragment sizes.Several methods that may obtain a source of artificially degraded DNA,including ultrasonication,DNase digestion and other physical methods(temperature and humidity,etc) have been reported so far,but there has not been a method accepted extensively.Therefore,we proposed to develop a method to prepare artificially degraded DNA under controlled conditions.
For the past few years,two new genetic markers have been developed,i.g. mini-STR and single nucleotide polymorphism(SNP).For forensic analysis of degraded DNA,it has become the essential strategy to reduce the amplicon length as short as possible.The typical technologies are mini-STR-PCR and mini-sequencing. However,the DNA fragment size should be more than 100～200bp when these two assays are used.If the average DNA fragment length is less than 100 bp,how can we get sufficient,reliable genetic information for forensic DNA analysis? It is still a big problem unsolved.
Nowadays,two main strategies are used in forensic DNA analysis of low-copy-number(LCN) samples:(1) enhancing STR-PCR assay.By increasing PCR cycle number,this method can give improved results.(2) nest PCR or whole genome amplification(WGA) combined with STR-PCR.After the use of WGA or nest PCR, the quantity of the starting DNA template increases greatly so that subsequent STR-PCR assay does not need much more cycle number.Because LCN samples only contain trace amounts of the starting DNA template,the stochastic effects will increase greatly,and result in allele imbalance and stutter product formation.In addition,it is often impossible to examine the LCN samples repetitively and difficult to assess its reliability.Therefore,the resulting profiles of both strategies above may be complex and difficult to interpret for forensic applications.
For the past decades,SNPs typing technologies based on ligase detection reaction (LDR) have developed quickly,and propose a new chance for forensic analysis of degraded DNA and trace DNA.First of all,LDR probes only need short target-specific sequences to hybridize to DNA template.The length of target-specific sequences should(1) be able to recognize the target locus to be tested,and(2) be suitable for recognition and binding by DNA ligase.Therefore,LDR probes can be as short as several dozens of nucleotides.In addition,LDR possesses high fidelity and stringency for base matching at the nick.Theoretically,LDR can be used for SNPs typing as long as the DNA fragment size is not less than 30～50bp.Thus,LDR is suitable for SNPs typing of highly degraded DNA.
After recognizing and hybridizing to target loci,padlock probes can be transformed circular molecules sealed by covalent bond.Reliable allelic discrimination achieves because of high fidelity and stringency of LDR.Subsequently,circularized padlock probes can be amplified for signal enhancement by hyper-branched circle amplification(HRCA).The amplification efficacy of HRCA is so exceeding that a great quantity of products can be obtained from trace DNA template.Therefore, LDR-HRCA may be valuable for SNPs typing of LCN DNA.
Methods
Twelve human peripheral blood samples(male 6,female 6) were provided by department of forensic Serology,forensic college,China Medical University.Genomic DNA was extracted by the standard phenol/chloroform procedure after digestion with proteinase K.
During the treatment of genomic DNA by ultrasonication or DNase digestion, aliquots were taken at a certain time-point,and the DNA was electrophoresed in agarose gel and amplified with the primers designed by ourselves.Therefore,the sizes of DNA fragments were clearly determined.
After preparation of the artificially degraded DNA,multiplex STRs loci were typed with AmpFl STR(?) Identifiler(?) PCR Amplification Kit.At the second step,four SNPs loci were selected(rs17750303,rs2307647,rs2307557,and rs17250992),and their LDR probes were designed.LDR probes were used to discriminate alleles by thermostable ligation reaction.Then,PCR was used to amplify the ligated LDR probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.
A series of LCN samples were prepared by diluting the starting DNA(500,100, 50,40,30,20,and 10 pg/μl,respectively).At the first step,AmpFl STR(?) Identifiler(?) PCR Amplification Kit was used to test the LCN samples.At the second step, Rs17750303 locus was selected,and allelic padlock probes(CPP and APP, corresponding to C-allele and A-allele respectively) were designed.Padlock probes were used to discriminate alleles by thermostable ligation reaction.Then,HRCA was used to amplify the circulized probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.
Results
1、After genomic DNA was ultrasonicated,the average sizes of DNA fragments varied significantly.The repeatability of ultrasonication was poor.As the digesting time extended,the average sizes of DNA fragments reduced gradually.After 1μg genomic DNA was digested with DNaseⅠfor 150 min,the fragment sizes of degraded DNA were not more than 100 bp.
2、Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit,no STR alleles could be obtained from the degraded DNA with fragment sizes less than 100bp.Using our LDR-PCR method,clear allelic discrimination of each target locus was achieved after staining of the final reaction mixtures with EB and visualization by UV illumination. The types of base variants did not influence the fidelity of DNA ligase.The target-specific sequence of LDR probes could be as short as 11 nucleotides,which did not disturb the ligation reaction.2 or 4 SNP loci could be correctly discriminated simultaneously in the same tube.Interaction between probes could be avoided easily when multiplex SNPs typing was done.
3、Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit and optimizing PCR conditions with 34 cycles,all STR loci could be discriminated when the starting DNA was not less than 100pg.Allele-drop occurred when the starting DNA was not more than 50pg.Using our LDR-HRCA method,final reaction products decreased as the amount of the starting genomic DNA decreased.Clear allelic discrimination was achieved as long as the homozygous DNA(AA or CC)was not less than 20 pg.10 samples of CC homozygosis(10pg of each) were detected.Among them,AC phenotype occurred in two,and no product in one.When 10 samples of AA homozygosis(10pg of each) were detected,AC phenotype occurred in one,CC phenotype in one,and no product in one.When heterozygous sample(AC) was detected,reliable allelic discrimination was achieved when the starting DNA was not less than 30 pg.10 samples of AC heterozygosis(10pg) were detected.CC phenotype occurred in one sample,AA phenotype in 2,and no product in one.No stutter was observed in all samples.
Conclusions
1、By treatment of genomic DNA with DNaseⅠ,it was reliable to control the degrees of DNA degradation.The method was able to be used to produce the heavily degraded DNA with a defined range of fragment lengths.
2、Using our LDR-PCR method,we successfully discriminated the genotypes of each target locus from highly degraded DNA with fragment sizes less than 100 bp. Meanwhile,multiplexed allelic discrimination of all 4 SNPs was achieved in a single tube.This LDR-PCR method might be valuable for forensic DNA analysis in those cases where DNA was damaged severely.
3、For DNA analysis of LCN,the specificity and sensitivity of our LDR-HRCA method were prior to enhancing STR-PCR assay,indicating that LDR-HRCA method might have potential in forensic applications.
4、Based on our study,we summarized the screening standards of target SNPs loci and designing principles of LDR probes.According to these principles,the specifity and sensitivity of SNPs typing were achieved when heavily degraded DNA or trace DNA was tested.In addition,high through-put SNPs typing could be achieved with the methods based on LDR.
最近二十年来法医物证学最引人注目的变革,就是DNA分析技术的应用及发展。对于高度降解检材和微量检材,如何从中获取可靠的、足量的遗传学信息,来进行法医学个体识别和亲权鉴定,一直是法医物证学面临的重大挑战。
随着降解DNA分析技术的不断发展,如何评价、比较不同方法的检验效能的问题,逐渐引起法医学者的关注;要实施此类研究,首先需要制备出特定片段长度的降解DNA标准样品。迄今,已提出了几种降解DNA的人工制备方法,包括超声振荡、DNA酶消化以及其它理化措施(改变环境温度、湿度、自然放置等),但是,这些方法目前均未被广泛接受。因此,本文尝试探讨高度降解检材的人工制备方法。
随着相关研究的不断深入,尽可能缩短靶DNA片段的扩增子长度,已经成为降解DNA分析的基本策略,目前mini-STR-PCR和mini-sequencing是其代表技术。但是,现有的mini-STR-PCR和mini-sequencing技术要求降解DNA片段的长度不得小于100bp～200bp。一旦基因组DNA发生了非常严重的降解,片段小于100bp的情况下,如何获得足量、可靠的遗传学信息来进行法医学鉴定呢?对此问题,目前尚无确切的解决方法。
当前,对于微量检材或低拷贝数检材(low copy number,LCN)的DNA分析,主要有两种检测策略:一是增强PCR-STR分型法,主要是增加PCR反应循环次数,提高STR位点扩增成功率(检测信号多寡)和产物量(检测信号的强弱),而后进行DNA分析;二是首先进行LCN检材的全基因组扩增或者巢式PCR扩增,以获取足量的DNA模板,而后进行DNA分型。但是,由于LCN检材所含DNA模板量过少,受随机效应的影响,采用上述两种策略,均会出现“额外”等位基因、影子带(stutter带)、等位基因丢失等一些难以避免的问题,STR分型结果通常具有不可重复性,并难以评价其有效性,无法得到可靠的法医学鉴定结论。
近年来,基于连接酶检测反应(LDR)的SNPs分型技术快速发展,为降解检材和微量检材的法医学DNA分析提供了新的契机。首先,LDR探针与模板DNA互补结合的序列不需过长,只要满足以下两个条件即可:(1)能够有效识别待测靶位点,(2)不影响连接酶的识别与结合所需片段长度,所以,LDR探针长度不超过数十个碱基。再者,对于双链DNA的单链切口,DNA连接酶的识别与连接反应具有高度严谨性。因此,从理论上说,只要DNA片段不小于30～50bp,即可采用LDR技术进行SNP分型检测,这一特性尤其适用于高度降解DNA分析。所以,本文尝试LDR-PCR策略来检测高度降解DNA。
再者,锁式探针(padlock probes)可识别靶位点并形成包含单链切口的局部双螺旋结构,经LDR反应可生成环状模板,LDR反应的高度严谨性保证了等位基因识别的准确性;同时,超分支滚环扩增反应具有极强的扩增效能,可极大地放大LDR检测信号。将二者结合起来,对LCN检材的分析效能将得以极大提高。所以,本文拟开发LDR-HRCA策略来检测LCN检材。
材料与方法
枸橼酸钠抗凝人末梢静脉血样品12份,男女各6份,由中国医科大学法医学院法医血清学教研室提供。采用酚—氯仿法提取基因组DNA。
分别使用超声法和DNaseⅠ酶切法处理高分子量基因组DNA,于不同时间段取样,电泳和/或PCR扩增检测DNA片段长度,人工制备不超过100bp的高度降解DNA检材。
使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit对降解DNA检材进行复合STR分型检验。选择4个待测SNPs位点(rs17750303、rs2307647、rs2307557和rs17250992),并构建各位点的LDR探针:首先采用LDR反应,识别降解检材各待测位点的基因型;而后采用PCR反应,扩增连接后LDR探针,根据PCR产物电泳结果判断各位点基因型。
使用稀释法制备DNA浓度梯度样品:200、100、50、40、30、20、10pg/μl。使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit对微量检材进行复合STR分型检验。选择rs17750303位点,并构建一对等位基因特异性锁式探针(CPP和APP);首先采用LDR反应,识别待测位点的基因型;而后采用HRCA反应,扩增环化锁式探针,根据电泳结果判断各位点基因型。
结果
1、超声法处理基因组DNA,可重复性不佳,DNA片段分布范围表现出明显差异。随着DNaseⅠ酶切时间延长,DNA片段长度不断缩短;酶切150min,降解DNA片段长度不超过100bp。
2、对于片段长度不超过100bp的人工降解DNA样品,AmpFl STR(?)Identifiler(?)PCR Amplification Kit中全部STR基因座均不能识别。采用LDR-PCR方法,进行单位点检测,单碱基置换变异(rs17750303和rs17250992)和三碱基缺失/插入变异(rs2307647和rs2307557)均能得到正确分型结果,多态性碱基的变异类型不是LDR反应忠实性的主要影响因素;短LDR探针(rs17750303和rs2307647)与长探针(rs2307557和rs17250992)均可得到准确分型结果,探针特异性互补序列可短至10个核苷酸而不影响LDR反应。进行2位点及4位点复合检测,均可获得准确分型结果,可有效避免探针间交互效应。
3、使用AmpFl STR(?)Identifiler(?)PCR Amplification Kit,PCR循环数34次,DNA模板量≥100ng,全部STR基因座均能正确识别;DNA模板量≤50ng,等位基因丢失现象严重,长扩增子座位尤为显著。采用LDR-HRCA方法检测rs17750303纯合型样品(CC型、AA型),产物量随DNA模板量降低而减少,20pg以上可获得正确的基因分型结果;CC纯合型样品10pg组共10份,CC表型7份,AC表型2份,无产物1份;AA纯合型样品10pg组共10份,AA表型6份,AC表型1份,CC表型1份,无产物2份。检测rs17750303杂合型样品(AC型),产物量随DNA模板量降低而减少,30 pg以上可获得正确的基因分型结果;10pg组共10份,AC表型6份,CC表型1份,AA表型2份,无产物1份。所有样品的检验过程中,均未发现影子带现象。
结论
1、采用DNaseⅠ酶切法处理基因组DNA,操作简单,DNA降解程度易于控制,结果稳定,可用于制备特定片段长度的降解DNA标准样品。
2、对于片段长度小于100bp的高度降解DNA检材,LDR-PCR方法可准确进行SNPs基因座分型,并可进行4个基因座复合分型检测。本方法对于高度降解检材的法医学DNA分析,具有较大的开发价值。
3、对于LCN检材,LDR-HRCA方法检测特异性好,灵敏度高于增强PCR-STR技术,可能更适用于法医学微量DNA分析。
4、本研究总结的待测SNPs位点筛选及相应LDR探针构建原则,可保证高度降解检材和微量检材SNPs分型的特异性和灵敏性,并且能够满足高通量检测的要求。
Background and objectives
For the past decades,the use of DNA analysis must be the most significant reformation in forensic fields.However,DNA degradation and trace DNA have always been special challenges for forensic scientists obtaining reliable and sufficient genetic information for personal identification and parentage testing.
To improve and assess the performance of different genotyping technologies in those cases where DNA is degraded,it would be desirable to have a reference sample of degraded DNA with known fragment sizes.Several methods that may obtain a source of artificially degraded DNA,including ultrasonication,DNase digestion and other physical methods(temperature and humidity,etc) have been reported so far,but there has not been a method accepted extensively.Therefore,we proposed to develop a method to prepare artificially degraded DNA under controlled conditions.
For the past few years,two new genetic markers have been developed,i.g. mini-STR and single nucleotide polymorphism(SNP).For forensic analysis of degraded DNA,it has become the essential strategy to reduce the amplicon length as short as possible.The typical technologies are mini-STR-PCR and mini-sequencing. However,the DNA fragment size should be more than 100～200bp when these two assays are used.If the average DNA fragment length is less than 100 bp,how can we get sufficient,reliable genetic information for forensic DNA analysis? It is still a big problem unsolved.
Nowadays,two main strategies are used in forensic DNA analysis of low-copy-number(LCN) samples:(1) enhancing STR-PCR assay.By increasing PCR cycle number,this method can give improved results.(2) nest PCR or whole genome amplification(WGA) combined with STR-PCR.After the use of WGA or nest PCR, the quantity of the starting DNA template increases greatly so that subsequent STR-PCR assay does not need much more cycle number.Because LCN samples only contain trace amounts of the starting DNA template,the stochastic effects will increase greatly,and result in allele imbalance and stutter product formation.In addition,it is often impossible to examine the LCN samples repetitively and difficult to assess its reliability.Therefore,the resulting profiles of both strategies above may be complex and difficult to interpret for forensic applications.
For the past decades,SNPs typing technologies based on ligase detection reaction (LDR) have developed quickly,and propose a new chance for forensic analysis of degraded DNA and trace DNA.First of all,LDR probes only need short target-specific sequences to hybridize to DNA template.The length of target-specific sequences should(1) be able to recognize the target locus to be tested,and(2) be suitable for recognition and binding by DNA ligase.Therefore,LDR probes can be as short as several dozens of nucleotides.In addition,LDR possesses high fidelity and stringency for base matching at the nick.Theoretically,LDR can be used for SNPs typing as long as the DNA fragment size is not less than 30～50bp.Thus,LDR is suitable for SNPs typing of highly degraded DNA.
After recognizing and hybridizing to target loci,padlock probes can be transformed circular molecules sealed by covalent bond.Reliable allelic discrimination achieves because of high fidelity and stringency of LDR.Subsequently,circularized padlock probes can be amplified for signal enhancement by hyper-branched circle amplification(HRCA).The amplification efficacy of HRCA is so exceeding that a great quantity of products can be obtained from trace DNA template.Therefore, LDR-HRCA may be valuable for SNPs typing of LCN DNA.
Methods
Twelve human peripheral blood samples(male 6,female 6) were provided by department of forensic Serology,forensic college,China Medical University.Genomic DNA was extracted by the standard phenol/chloroform procedure after digestion with proteinase K.
During the treatment of genomic DNA by ultrasonication or DNase digestion, aliquots were taken at a certain time-point,and the DNA was electrophoresed in agarose gel and amplified with the primers designed by ourselves.Therefore,the sizes of DNA fragments were clearly determined.
After preparation of the artificially degraded DNA,multiplex STRs loci were typed with AmpFl STR(?) Identifiler(?) PCR Amplification Kit.At the second step,four SNPs loci were selected(rs17750303,rs2307647,rs2307557,and rs17250992),and their LDR probes were designed.LDR probes were used to discriminate alleles by thermostable ligation reaction.Then,PCR was used to amplify the ligated LDR probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.
A series of LCN samples were prepared by diluting the starting DNA(500,100, 50,40,30,20,and 10 pg/μl,respectively).At the first step,AmpFl STR(?) Identifiler(?) PCR Amplification Kit was used to test the LCN samples.At the second step, Rs17750303 locus was selected,and allelic padlock probes(CPP and APP, corresponding to C-allele and A-allele respectively) were designed.Padlock probes were used to discriminate alleles by thermostable ligation reaction.Then,HRCA was used to amplify the circulized probes for signal enhancement.The genotypes of each locus were identified according to results of gel electrophoresis.
Results
1、After genomic DNA was ultrasonicated,the average sizes of DNA fragments varied significantly.The repeatability of ultrasonication was poor.As the digesting time extended,the average sizes of DNA fragments reduced gradually.After 1μg genomic DNA was digested with DNaseⅠfor 150 min,the fragment sizes of degraded DNA were not more than 100 bp.
2、Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit,no STR alleles could be obtained from the degraded DNA with fragment sizes less than 100bp.Using our LDR-PCR method,clear allelic discrimination of each target locus was achieved after staining of the final reaction mixtures with EB and visualization by UV illumination. The types of base variants did not influence the fidelity of DNA ligase.The target-specific sequence of LDR probes could be as short as 11 nucleotides,which did not disturb the ligation reaction.2 or 4 SNP loci could be correctly discriminated simultaneously in the same tube.Interaction between probes could be avoided easily when multiplex SNPs typing was done.
3、Using AmpFl STR(?) Identifiler(?) PCR Amplification Kit and optimizing PCR conditions with 34 cycles,all STR loci could be discriminated when the starting DNA was not less than 100pg.Allele-drop occurred when the starting DNA was not more than 50pg.Using our LDR-HRCA method,final reaction products decreased as the amount of the starting genomic DNA decreased.Clear allelic discrimination was achieved as long as the homozygous DNA(AA or CC)was not less than 20 pg.10 samples of CC homozygosis(10pg of each) were detected.Among them,AC phenotype occurred in two,and no product in one.When 10 samples of AA homozygosis(10pg of each) were detected,AC phenotype occurred in one,CC phenotype in one,and no product in one.When heterozygous sample(AC) was detected,reliable allelic discrimination was achieved when the starting DNA was not less than 30 pg.10 samples of AC heterozygosis(10pg) were detected.CC phenotype occurred in one sample,AA phenotype in 2,and no product in one.No stutter was observed in all samples.
Conclusions
1、By treatment of genomic DNA with DNaseⅠ,it was reliable to control the degrees of DNA degradation.The method was able to be used to produce the heavily degraded DNA with a defined range of fragment lengths.
2、Using our LDR-PCR method,we successfully discriminated the genotypes of each target locus from highly degraded DNA with fragment sizes less than 100 bp. Meanwhile,multiplexed allelic discrimination of all 4 SNPs was achieved in a single tube.This LDR-PCR method might be valuable for forensic DNA analysis in those cases where DNA was damaged severely.
3、For DNA analysis of LCN,the specificity and sensitivity of our LDR-HRCA method were prior to enhancing STR-PCR assay,indicating that LDR-HRCA method might have potential in forensic applications.
4、Based on our study,we summarized the screening standards of target SNPs loci and designing principles of LDR probes.According to these principles,the specifity and sensitivity of SNPs typing were achieved when heavily degraded DNA or trace DNA was tested.In addition,high through-put SNPs typing could be achieved with the methods based on LDR.
引文
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