A Small Characterised Human Genome Library

Project leader: Raymond Dalgleish.

This project was funded by the GENIE CETL grant.

For several years we have taught aspects of human genome organisation to second-year undergraduate students by way of a series of laboratory experiments in which they analyse a small library of genomic DNA fragments using a plasmid vector.

In the modern era of molecular genomics it became apparent that an element of bioinformatics needed to be incorporated into this exercise. A new library of human DNA fragments has been created in the plasmid vector pUC19 by ligation of human DNA partially digested with Sau3AI into the unique BamHI site of the vector.

pUC19 Vector

Around 90 clones were selected and have had their DNA sequence determined at each end of the human DNA insert, including the junction between the vector and the insert.

  • Students are assigned a unique individual clone.
  • They isolate plasmid DNA from a bacterial culture harbouring their clone and analyse the DNA by restriction enzyme digestion and by PCR amplification of the insert using vector-specific primers.
  • The amplification products are electrophoresed & Southern blotted and hybridised with a probe capable of detecting repetitive Alu elements.
  • From this analysis the students are able to say if their clone harbours an Alu element, but not how many as the process is not quantitative.
  • Using the DNA sequence data, the students are first asked to analyse the junction between the vector and insert to determine whether or not BamHI sites were re-created during the ligation reactions.
  • Next, they localise their cloned segment of human DNA to a specific chromosome band using the USCS Genome Browser.
  • Finally, they are also able to precisely determine the size of the human DNA insert and how many Alu elements (if any) are actually present.

DNA Trace

Future Work:

Some additional analysis of the clone library is continuing.

Publications:

An account of this project has been published in Journal of Biological Education:

Dalgleish R , Shanks ME, Monger K & Butler NJ (2012) A mini-library of sequenced human DNA fragments: linking bench experiments with informatics. Journal of Biological Education 46(3), 193-198.

Copies of this paper are available on request to those without direct access to the journal.

Availability as an OER:

The bacterial clones containing the human DNA inserts and their associated sequence data are being made available as an open educational resource (OER). Please get in contact if you wish to incorporate this resource into your teaching programme.

 

 

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