应用SSH-PCR技术进行混合血样STR分型的初步研究

发布时间：2018-03-29 20:57

本文选题：法医物证学　切入点：低含量组分　出处：《中国医科大学》2010年硕士论文

【摘要】： 混合斑是指两名或两名以上个体的体液、分泌液混合形成的斑痕。可以分为两类：一类是由不同个体的同一种体液、分泌液混合而成,最常见为混合血痕；另一类是由不同个体的不同体液、分泌液混合而成,最常见为精液与阴道分泌液组成的混合斑。一个样本中多个DNA来源个体的检出有赖于每个个体DNA在样本所占的比例,基因型的组合,及待扩增样本的DNA总量,即混合样本DNA分型的难易程度不尽相同。混合样本中较少成分所占的比例小于5%或1：19,通常是无法检测到。AmpF1STR试剂盒建议,混合样本中最少的成分应该大于35pg,才能得到可靠的分型结果。提高混合样本中较少组分DNA分型成功率,是法医工作者急需解决的难题之一,同时在医学遗传学领域也具有重要的研究价值。SSH-PCR (Suppression Subtractive Hybridization SSH)是1996年Diatchenko等以抑制性PCR为基础、并结合标准化与消减杂交技术,建立的一项筛选与分离未知差异表达基因的新技术。其基本过程是：抽提两种不同细胞的差异mRNA,反转录成cDNA,再用限制性内切酶消化cDNA成平末端片段,将待研究的cDNA连接接头后作为检测子,将另一cDNA作为驱动子进行两轮杂交,最后通过两次PCR扩增得到差异表达的cDNA。该方法运用了杂交二级动力学原理,即丰度高的单链DNA在退火时产生同源杂交的速度快于丰度低的单链DNA,从而使原来在丰度上有差别的单链DNA相对含量达到基本一致。而抑制PCR则是通过利用链内退火优于链间退火的特点,使两端接上具有反向末端重复序列的同一接头的非目片段在退火时产生类似发夹的互补结构,无法作为模板与引物配对,从而选择性地抑制非目的基因片段的扩增。在标准体系中,运用SSH进行一轮消减杂交,可富集稀有序列1000倍以上,使某些低丰度表达的mRNA有望被检出。鉴于SSH技术富集差异DNA序列的能力,本研究拟以PCR-STR扩增产物为研究对象,直接连接接头,并通过消减杂交和抑制PCR扩增,提高混合检材中较少组分DNA的分型成功率,为混合样本的DNA分析提供一种新的方法。材料和方法 1、血液样品由中国医科大学法医血清教研室提供。 2、不同型别血液样品分别按照1：3、1：5、1：7、1：9、1：19、1：29、1：39混合制成。结果 1、不同型别血液样品分别按照1：3、1：5、1：7、1：9、1：19、1：29、1：39混合后聚丙烯酰胺凝胶电泳检测发现,当混合比例高于1：9时,在凝胶显色后无法观察到明显的条带。这表明用聚丙烯酰胺凝胶电泳的方法不如毛细管电泳法灵敏,其检出的极限比列明显低于毛细管电泳法的1：19。 2、使用不同浓度的T4 DNA连接酶进行连接后检测,当酶浓度为35U/μl(相当于0.28Weiss单位)时,连接效果明显而使用其他浓度连接酶进行连接反应,其检测结果不明显,在预期位置未发现有明显条带。 3、第二次杂交产物聚丙烯酰胺凝胶电泳检测结果,高含量组分被明显抑制,在预期位置未发现有明显条带(183bp+22bp+20bp=225bp),低含量组分得到放大,在预期位置(171bp+22bp+20bp=213bp)出现明显条带。 4、不同杂交条件下,对其杂交产物进行扩增并检测出现不同的结果。在预期位置(213bp)未出现明显条带。在300bp以上各泳道均出现不同强度的条带。结论应用SSH-PCR技术进行混合样本检测时,当混合比例1：9时将高含量组分抑制,低含量组分被有效放大。
[Abstract]:Mixed plaque refers to the liquid and secretion of two or more individuals . It can be divided into two types : one is composed of the same kind of body fluid and secretion of different individuals , the most common is mixed bloodstain ;
A new technique for screening and isolating unknown differentially expressed genes was established by using two - stage hybridization technique .

In view of the ability of SSH to enrich the differential DNA sequence , the PCR - STR amplification product was used as the research object to directly connect the linker , and the success rate of the less component DNA in the mixed sample was improved by reducing the hybridization and inhibiting the PCR amplification , thus providing a new method for DNA analysis of the mixed sample .

Materials and Methods

1 . Blood samples are provided by the doctor ' s serum teaching and research laboratory of Chinese Medical University .

2 . The blood samples of different types were mixed with 1 : 3 , 1 : 5 , 1 : 7 , 1 : 9 , 1 : 19 , 1 : 29 , 1 : 39 , respectively .

Results

The results showed that the polyacrylamide gel electrophoresis was not as sensitive as capillary electrophoresis , and the detection limit ratio was significantly lower than that of capillary electrophoresis .

2 . After ligation with T4 DNA ligase at different concentrations , when the enzyme concentration was 35U / 渭l ( equivalent to 0.28weer unit ) , the ligation effect was obvious and the ligation reaction was carried out using other concentration ligase . The results were not obvious , and no obvious bands were found in the expected position .

3 . The results of polyacrylamide gel electrophoresis in the second hybridization showed that the high content components were obviously inhibited , and no obvious bands were found in the expected position ( 183bp + 22bp + 20bp = 225 bp ) , and the low content components were amplified , and there appeared obvious bands in the expected position ( 171bp + 22bp + 20bp = 213bp ) .

4 . The hybridization products were amplified and detected under different hybridization conditions . There were no obvious bands in the expected position ( 213bp ) . bands of different intensities appeared in each lane above 300 bp .

Conclusion

When mixed sample detection is performed using SSH - PCR technology , the high content component is suppressed and the low content component is effectively amplified when the mixing ratio is 1 : 9 .

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919

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