A Research On Strategy For Amplifying And Sequencing Complex Regions Of The Human Genome

Although the Human Genome Project was ?nished in 2004, the sequence of thehuman genome is not perfect and there are still hundreds of sequencing gaps withdi?erent sizes. Five years passed, these gaps still remain. Today, people still havelimited knowledge of their characteristic. It is generally believed that the gaps havecomplex sequence structures and there are multiple obstacles, such as low ampli?-cation e?iciency, low ampli?cation speci?city, hard-to-clone ampli?cation products,when amplifying these regions with polymerase chain reaction (PCR). They can notbe solved by current technologies and closing these gaps requires more research worksand new technology improvements. As an emerging nano-biotechnology, nanoparti-cle PCR was discovered to have multiple optimizing e?ects on PCR compared withtraditional methods, which may be a potential solution.Here, a sequencing gap in human chromosome 5 was chosen as the research sys-tem. Combining nanoparticle PCR and DNA atomic force microscopy with molecularbiological methods such as nested-PCR, molecular cloning, this gap was ampli?ed,sequenced and analyzed, and ?nally the complete sequence was got.The results showed that:1. Because of the extraordinary complexity of this ampli?cation system, thenanoparticle PCR had no evident optimizing e?ects when used alone. However, com-bined with nested-PCR, it could enhance the ampli?cation e?iciency and speci?cityof nested-PCR which was insu?icient when amplifying this complex sequence region.The enhancement of speci?city made the sequencing give higher base quality values. This feature played a key role in getting partial sequence of this gap which was thebasis of the following work.2. An extreme complex region was found in this gap, leading to: a. Ampli?edproduct length polymorphism was observed when amplifying this region with variousprimer pairs; b. Some of the ampli?cation products had inconsistent sequencing resultafter cloning, the length of sequence was obviously shorter than the original fragment;c. The fragment which covered this whole region had very low cloning e?iciency;d. Some of the ampli?cation products formed a cruciform secondary structure. Theanalysis on the ?nal sequence of this gap proved that this region was rich in bothforward and reverse repeats, and the coactions of the two types of repeats led tosegment loss during ampli?cation and ampli?ed product length polymorphism. These?ndings explained why this gap remained and was di?icult to solve, helped us to knowmore about the characteristic of the gaps, and shed light on developing more perfectstrategy for amplifying and sequencing complex regions of the human genome.There are still many problems to solve in these most intractable parts in humangenome sequencing. Here, combining multiple nanotechnologies and biotechnologies,an e?ective method that can partially solve the problems in closing human genomesequencing gaps was proposed.