An optimized ligation-mediated PCR method for chromosome walking and fusion gene chromosomal breakpoints identification

Author:

Lung Jrhau123ORCID,Hung Ming-Szu245,Chen Chao-Yu678,Yang Tsung-Ming4,Lin Chin-Kuo4,Fang Yu-Hung45,Jiang Yuan-Yuan4,Liao Hui-Fen9,Lin Yu-Ching245

Affiliation:

1. Department of Medical Research and Development, Chang Gung Memorial Hospital , Chiayi, 613, Taiwan

2. Department of Medicine, College of Medicine, Chang Gung University , Taoyuan, 333, Taiwan

3. Center for General Education, Chang Gung University of Science and Technology, Chiayi Campus , Chiayi, 613, Taiwan

4. Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital , Chiayi, 613, Taiwan

5. Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus , Chiayi, 613, Taiwan

6. Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital , Chiayi, 613, Taiwan

7. Department of Early Childhood Care and Education, Shu-Zen Junior College of Medicine and Management , Kaohsiung, 821, Taiwan

8. Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University , Taoyuan, 333, Taiwan

9. Department of Biochemical Science and Technology, National Chiayi University , Chiayi, 600, Taiwan

Abstract

Abstract Molecular techniques that recover unknown sequences next to a known sequence region have been widely applied in various molecular studies, such as chromosome walking, identification of the insertion site of transposon mutagenesis, fusion gene partner, and chromosomal breakpoints, as well as targeted sequencing library preparation. Although various techniques have been introduced for efficiency enhancement, searching for relevant single molecular event present in a large-sized genome remains challenging. Here, the optimized ligation-mediated polymerase chain reaction (PCR) method was developed and successfully identified chromosomal breakpoints far away from the exon of the new exon junction without the need for nested PCR. In addition to recovering unknown sequences next to a known sequence region, the high efficiency of the method could also improve the performance of targeted  next-generation sequencing (NGS).

Funder

Chang Gung Memorial Hospital, Chiayi, Taiwan

Publisher

Oxford University Press (OUP)

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