用PCR和ESI-TOF-MS技术检测线粒体DNA的异质性

目的用PCR和ESI-TOF-MS分型技术检测线粒体DNA（mtDNA）D环高变区,通过碱基组成分析mtDNA的异质性。方法从华东汉族群体选取12名无关个体,用PLEX-ID平台进行mtDNA分型。该平台使用12对引物,对mtDNA高变区1（HVⅠ,引物所跨区域为15893~16451）进行碱基组成分析;使用另外12对引物,对mtDNA高变区2（HVⅡ,引物所跨区域为5~603）进行碱基组成分析,考察mtDNA异质性频率。结果 mtDNA多态性区域的碱基组成信息反映出区段内有无异质性。在高变区Ⅰ的12个区段中,有3个区段表现出多聚C长度异质性：在mtDNA高变区Ⅱ（31~576）的12个区段中,有3个区段检见点异质性,另外5个区域检见Poly C长度异质性。结论群体调查表明,mtDNA的序列异质性多见于高变区Ⅱ的103~267区段,多聚C长度异质性多见于高变区Ⅰ的16124~16201、16157~16201、16182~16250区段和高变区Ⅱ的234~367、431~576区段。将mtDNA标记用于母系关系检验和（或）个体识别时,需要格外留意这些异质性信息,以免结论错误。     

线粒体DNA异质性在法医遗传学中的研究进展及存在的问题探讨

1 概述 人类mtDNA 1981年在英国剑桥Sanger实验室首次完成全序列测定,这个最初测定的序列(基因库编码:M63933)作为对比的参考序列,通常被称为Anderson序列或剑桥序列[1].线粒体DNA(mitochondrial DNA,mtDNA)为环状DNA,有16569碱基对,含37个编码氧化磷酸化过程相关物质的基因,还有一个复制控制区称为D-环区.该区在个体间呈现多态性,可用于人类个体识别和亲子鉴定.D-环区包括三个高变区(hypervariable region,HV)目前已有人提出高变区Ⅳ的概念,但文献报道较少.无血缘关系个体中mtDNA的HVⅠ和HVⅡ区域变化率大约在1%～3%[2].     

mtDNA异质性及其法医学应用

线粒体DNA（mitochondrial DNA,mtDNA）是惟一的细胞核外DNA.人类mtDNA为一裸露的环状双链结构,长16569 bp,由富含嘌呤的重链（H链）和富含嘧啶的轻链（L链）组成.mtDNA编码区的序列相对保守,其非编码区长1122bp,又称为控制区.控制区的碱基变异相对集中分布在15996～16401nt和29～408nt两个区段,分别称为HV Ⅰ和HVⅡ.后来,Lutz等发现在438～574nt间也存在较多的碱基变异,称为HVⅢ.mtDNA呈母系遗传特征,加之其拷贝数多、突变率高、抗腐败能力强,具有极高的法医学应用价值.     

线粒体DNA编码区的多态性

目的研究线粒体DNA(mtDNA)编码区单核苷酸多态性,建立mtDNA编码区多态性在法庭科学中应用的理论基础。方法针对mtDNA编码区nt8162-8483以及nt13070-13299两段序列设计引物,应用直接测序技术研究其多态性。结果两对引物扩增片段长分别为322bp和230bp,共检测到21种变异,24种单倍型,基因多样性为0.7511,两个无关个体的偶合概率为0.2564。结论线粒体DNA编码区多态性位点作为线粒体DNA控制区多态性位点的补充,联合应用可以提高线粒体DNA在法医学应用中的个体识别能力。     

中国广东汉族群体mtDNA控制区的多态性

目的 探讨线粒体DNA控制区(包括HVⅠ区、HVⅡ区和HVⅢ区)的多态性。方法 采用PCR扩增和末端标记荧光循环测序的方法，对100名广东汉族无关个体进行了序列分析。结果 共观察到147个变异位点，序列变异包括了碱基转换、颠换、插入、缺失等各种类型。其中在HV Ⅰ区(nt16，024～nt16，365)内观察到88个变异位点，91种单倍型，基因多样度为0.9964；在HVⅡ区(nt73～nt340)观察到42个变异位点，67种单倍型，基因多样度为0.9861；在HVⅢ区(nt438～nt574)观察到9个变异位点，15种单倍型，基因多样度为0.8760。联合3个高变区域的序列，可观察到98种单倍型，基因多样度为0.9996。结论 本研究为线粒体DNA在法庭科学中的应用提供了较系统的实验依据。结果还表明，对于mtDNA等单倍型遗传标记，增加其检测范围，可提高该系统的个体识别能力，使其在法庭科学领域充分发挥作用。     

利用线粒体DNA异质性检验碎尸案1例

线粒体DNA(mtDNA)异质性在法医学鉴定中具有双重价值,当比对的检材中只有一份存在mtD-NA异质性时,给法医学鉴定增加了不确定性,但当比对的检材在相同的位置均出现相同的异质性时,其异质性可作为特殊的遗传标记增加证据的强度。本文就在实际案件检验中遇到的在相同位置均存在长度异质型作一报道。1案件简介2005年12月2日在北京市海淀区某河床发现尸块,同时在现场提取到枕巾上附着的毛发,毛发已无毛囊,失去STR检验条件。因此对其进行mtDNA高变区Ⅰnt16030-16481和高变区Ⅱnt15-484扩增测序,并与尸块血痕进行比对。结果发现毛发及尸块血…     

四类遗传标记的检测用于混合精斑的个人识别

目的探讨综合应用常染色体STR与Y-STR、X-STR、mtDNA高变区Ⅰ序列检测四类遗传标记,提高两男性混合精液样品的个人识别率。方法使用商品化试剂盒检测混合精液样品的常染色体与X、Y染色体STR,应用单链构象多态性(single strand conformation polymorphism,SSCP)法分离两男性混合精液样品的线粒体DNA(mitochondrial DNA,mtDNA)高变区Ⅰ序列,并进行序列分析。结果混合精液样品比例超过1:10(高组分所占比例继续增加而低组分所占比例继续减少)时可以确定高拷贝者的全部四类遗传标记系统的分型。比例接近时,通过与嫌疑人检测结果的比较可获得排除或基本认定的意见。结论四类遗传标记系统的联合检测有助于提高两男性混合精液样品的个人识别率。     

多种遗传标记在同母异父半同胞关系鉴定中的综合应用

目的通过对常染色体和X染色体STR基因座以及线粒体DNA高变区多态性的检验,探讨同母异父半同胞关系鉴定策略。方法提取3名全同胞及其1名疑似同母异父半同胞个体的DNA,采用SinofilerTM试剂盒检测常染色体上的15个STR基因座、采用Mentype Argus X-8试剂盒和自主研制的16重X染色体STR扩增体系,共检验X染色体上的19个STR基因座,同时采用基因测序技术分析线粒体DNA高变区Ⅰ和高变区Ⅱ的多态性。结果依据常染色体STR基因型结果及全同胞指数和半同胞指数计算结果排除可疑个体与已知3名全同胞间存在全同胞关系,线粒体DNA高变区多态性检测结果提示4名被鉴定人为同一母系,X染色体STR分型结果支持被鉴定个体间为半同胞关系。结论对于同母异父半同胞鉴定案例,综合运用常染色体STR、X染色体STR及线粒体DNA高变区测序等多种遗传分析手段,可获得可靠的鉴定结论。     

线粒体DNA的PCR-RFLP分型法医学应用研究

目的建立PCR RFLP技术检测mtDNA序列多态性的方法,调查武汉汉族人群mtDNAHVⅠ区段限制性片段长度多态性,并对PCR RFLP技术在毛干、指甲等生物检材的个体识别案件中的应用进行评估。方法应用nest PCR技术和RFLP技术建立检测mtDNAHVⅠ区段限制性片段长度多态性的方法,调查150例武汉汉族人群无关个体,同时对实际案件中的毛干、陈旧骨骼、水浸血痕等不同种类、不同保存时间和条件的生物检材进行检测。结果RsaⅠ酶切检出4种表型,频率分别为0.760、0.167、0.066和0.007,遗传差异度(GD值)为0.393,随机匹配概率(P值)为0.607。应用PCR RFLP对毛干、20年陈旧骨骼、水浸血痕进行了检测以及在个体识别及母子关系亲子鉴定案例中应用。结论用PCR RFLP法检测mtDNA序列多态性在法医物证检验中具有应用价值。     

线粒体高变区多聚C-stretch序列长度多态性

目的研究线粒体高变区多聚C-stretch序列长度多态性,并探讨其在法医学个体识别中的价值。方法针对线粒体高变区nt16180及nt310两个位点采用文献报道引物,应用直接测序技术研究其等位基因分布及频率。结果两对引物扩增长度分别为807bp和962bp,nt16180位点检测到7种基因型,其中AAAACCCCCTCCCC基因型占87.72%,AAAACCCCCCCCCCCCC基因型在汉族人群中首次报道;nt310检测到7种基因型,其中CCCCCCCCTCCCCCC基因型占60.53%;联合两个位点共检测出15种单倍型,GD值为0.6309,其中AAAACCCCCTCCCC-CCCCCCCCTCCCCCC检测出66条,达到57.89%。结论为线粒体控制区DNA在法医学领域中的应用提供基础数据,证实了线粒体nt16180位点和nt310位点单倍型在线粒体DNA鉴定中有较好的应用价值。     

Sequencing strategy of mitochondrial HV1 and HV2 DNA with length heteroplasmy

We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2-4 bases downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mtDNA region is preferable in order to reduce ambiguities in sequence determination.     

中国汉族人群的线粒体DNA控制区多态性研究

探讨mtDNA多态性在法庭科学中个体识别的理论基础。应用PCR扩增产物直接测序方法 ,对 111名中国北方地区汉族人群无血缘关系个体的mtDNA控制区 (HVⅠ和HVⅡ )进行测序分析。在高变区Ⅰ 15 998～ 16 40 0之间发现 10 2处碱基变异 ,10 3个mtDNA单倍型 ;在高变区Ⅱ 0 0 0 35～ 0 0 36 9之间的发现 36处碱基变异 ,6 9个mtDNA单倍型。其可变碱基的变异形式主要为碱基替代 (转换和颠换 )、插入和缺失 ;碱基转换 (78 9% )明显高于颠换(14 3% )、插入 (3 4% ) ,缺失 (3 4% )。分析表明 ,人群个体mtDNA控制区碱基序列 ,基因多样性为 99 9% ,两个无关个体的偶合概率为 0 92 % ,具有高度序列的多态性     

Mitochondrial DNA control region polymorphism in the population of Alagoas state, north-eastern Brazil

The sequences of the two hypervariable (HV) segments of the mitochondrial DNA (mtDNA) control region were determined in 167 randomly selected, unrelated individuals living in the state of Alagoas, north-eastern Brazil. One hundred and forty-five different haplotypes, associated with 139 variable positions, were determined. More than 95% of the mtDNA sequences could be allocated to specific mtDNA haplogroups according to the mutational motifs. Length heteroplasmy in the C-stretch HV1 and HV2 regions was observed in 22 and 11%, respectively, of the population sample. The genetic diversity was estimated to be 0.9975 and the probability of two random individuals presenting identical mtDNA haplotypes was 0.0084. The most frequent haplotype was shared by six individuals. All sequences showed high-quality values and phantom mutations were not detected. The diversity revealed in the mitochondrial control region indicates the importance of this locus for forensic casework and population studies within Alagoas, Brazil.     

Identification of remains by sequencing of mitochondrial DNA control region

The maternity of two newborns who were murdered and abandoned >5 and 10 years were analyzed by amplification and direct sequencing of mitochondrial DNA (mtDNA) control regions. Sequences of two hypervariable segments from each femur bone sample and the blood of the putative mother showed four mutations in hypervariable region I and two mutations in addition to two nucleotide insertions in hypervariable region II compared with the reference sequence, and all sequences were identical. The genotype of these individuals is found to be relatively rare in the Japanese population, and it was strongly suggested that both sets of newborn remains really were children of the putative mother. Sexes of the remains were determined to be female and male by amplifying a segment of the X-Y homologous gene, amelogenin. These results demonstrate that sequencing of mtDNA is a useful tool for genetic identification of aged and decomposed materials.     

A database of mitochondrial DNA hypervariable regions I and II sequences of individuals from Slovakia

In order to identify polymorphic positions and to determine their frequencies and the frequency of haplotypes in the human mitochondrial control region, two hypervariable regions (HV1 and HV2) of the mitochondrial DNA (mtDNA) of 374 unrelated individuals from Slovakia were amplified and sequenced. Sequence comparison led to the identification of 284 mitochondrial lineages as defined by 163 variable sites. Genetic diversity (GD) was estimated at 0.997 and the probability of two randomly selected individuals from population having identical mtDNA types (random match probability, RMP) for the both regions is 0.60%.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High-Performance Liquid Chromatography

Abstract:  Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.