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[Isolating candidate inserted fragment from positive fused phage clones using quantitative PCR].
1Institute of Genetics,Fudan University, Shanghai,200433 P.R. China. Longyu@fudan.edu.cn
Summary
This study presents a rapid and precise method for isolating specific gene fragments from fused phage clones using quantitative polymerase chain reaction (PCR). This technique efficiently identifies the HumGT-H1 gene
Area of Science:
- Molecular Biology
- Gene Cloning
- Recombinant DNA Technology
Context:
- Fused phage clones are often used to construct cDNA libraries.
- Identifying specific inserted fragments within these clones can be challenging and time-consuming.
- Traditional methods like hybridization screening may require multiple steps.
Purpose:
- To develop a rapid and exact method for isolating specific cDNA fragments from fused phage clones.
- To avoid the need for secondary hybridization screening.
- To efficiently identify the 5' cDNA sequence of the HumGT-H1 gene.
Summary:
- A novel method utilizing quantitative polymerase chain reaction (PCR) was employed to isolate target cDNA fragments based on template quantity differences.
- This two-PCR approach successfully identified a specific 1.9kb 5' cDNA fragment of the HumGT-H1 gene from a two-phage fused clone.
- The method eliminates the necessity for subsequent hybridization screening.
Impact:
- Provides an effective and rapid gene cloning technique.
- Significantly reduces time and material costs in molecular biology research.
- Facilitates faster identification and characterization of cloned genes.