Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Optimization of long-distance PCR using a transposon-based model system.

L D Ohler1, E A Rose

  • 1Human Genome Laboratory, Perkin-Elmer Cetus Instruments, Emeryville, California 94608.

PCR Methods and Applications
|August 1, 1992
PubMed
Summary

Researchers developed a new method for amplifying long DNA fragments (up to 9 kb) using transposon-based PCR. This technique improves specificity and fidelity for genome mapping and sequencing applications.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

The effect of prescribed and preferred intensity exercise on psychological affect and the influence of baseline measures of affect.

Journal of health psychology·2011
Same author

Exercise experience influences affective and motivational outcomes of prescribed and self-selected intensity exercise.

Scandinavian journal of medicine & science in sports·2010
Same author

Destination mechanical circulatory support: proposal for clinical standards.

The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation·2003
Same author

Massive air embolism with left ventricular assist device.

Neurology·2002
Same author

Long-term use of a left ventricular assist device for end-stage heart failure.

The New England journal of medicine·2002
Same author

Multicenter clinical evaluation of the HeartMate vented electric left ventricular assist system in patients awaiting heart transplantation.

The Journal of thoracic and cardiovascular surgery·2001

Area of Science:

  • Molecular Biology
  • Genomics

Background:

  • Standard PCR is limited to amplifying DNA fragments <3 kb.
  • Generating long PCR products (5-25 kb) with high specificity is challenging.
  • Existing methods for long PCR products are often difficult to replicate.

Purpose of the Study:

  • To develop and evaluate methodological changes for efficient long-range PCR.
  • To enable routine amplification of long PCR products (5-25 kb) with high specificity and fidelity.
  • To benefit genome mapping and sequencing endeavors.

Main Methods:

  • Utilized a model system with transposon Tn5supF to introduce primer binding sites.
  • Employed transposon-based PCR to study amplification of DNA fragments with varying size and sequence.
  • Optimized reaction conditions using recombinant Thermus thermophilus (rTth) DNA polymerase, gelatin, wax-mediated hot starts, and auto-segment extension thermocycling.

Related Experiment Videos

Main Results:

  • Achieved specific PCR amplification of DNA templates up to 9 kb.
  • Demonstrated the effectiveness of the developed methodology for long-range PCR.
  • Identified key methodological features enabling efficient amplification of long DNA fragments.

Conclusions:

  • The described conditions enable PCR amplification of specific DNA templates up to 9 kb.
  • The methodology offers a reliable approach for generating long PCR products.
  • Provides insights for further enhancement of long-distance PCR capabilities.