Related Experiment Video
Updated: Jul 17, 2026
09:31
Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Published on: March 22, 2016
Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.
Yue-Hua Xiao1, Yi Peng, Ming Luo
1Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400716, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao = Journal of Plant Physiology and Molecular Biology
|February 9, 2007
Summary
The Y-shaped adaptor dependent extension (YADE) method for DNA amplification is improved by using multiple templates. This enhanced YADE approach increases efficiency for sequential amplification of flanking sequences in complex genomes.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- The Y-shaped adaptor dependent extension (YADE) method is valuable for amplifying flanking DNA sequences.
- YADE's efficiency can be restricted by the presence of specific restriction sites near the target sequence.
Purpose of the Study:
- To enhance the efficiency of the YADE method for amplifying flanking DNA sequences.
- To adapt YADE for sequential amplification of unknown genomic regions.
Main Methods:
- Developed a modified YADE approach utilizing multiple DNA templates.
- Generated templates through multiple restriction and ligation steps.
- Applied sequential YADE amplifications to amplify a 5'-upstream sequence of a cotton gene.
Main Results:
- The multiple-template strategy significantly increased YADE method efficiency.
- Successfully amplified a 2,228-bp 5'-upstream sequence of a cotton small GTPase gene.
- Achieved amplification through two rounds of sequential YADE using templates from 7 digestions.
Conclusions:
- The multiple-template YADE method is effective for overcoming limitations posed by restriction sites.
- This modified YADE technique is suitable for sequential PCR walking in complex genomes.
- Offers a robust tool for exploring flanking sequences in genomic research.
Related Concept Videos
02:10Translesion DNA Polymerases
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
03:01The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
02:35RACE - Rapid Amplification of cDNA Ends
Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...
Since the...
03:00Next-generation Sequencing
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
01:32PCR
Overview
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...