Attempting to make a genomic library of an extinct animal

The Australian Museum's research in its attempt to clone a thylacine has focussed on the strategy of constructing a genomic library of this extinct animal.

Project Goals

The immediate goals at the commencement of the project were to:

  1. Extract DNA of the highest possible quality from thylacine specimens.
  2. Make and distribute "libraries" in bacteria or yeast, with complete coverage of the thylacine genome so that its genetic material can be maintained indefinitely.

DNA Extraction

Early work on the project focussed on extracting DNA from the ethanol preserved thylacine pouch young (specimen P762). Approximately 200 nanograms of liver DNA with an average size of between 1200 and 2000 bases was extracted. This was too little to visualise on an ordinary DNA gel (Figure 1) but could be visualised and quantified by making the DNA radioactive (by "end-labelling") (Figure 2). Unfortunately subsequent tests ("PCR amplification") showed that whilst there was some thylacine DNA in the extraction, the majority of the sample was from contaminating micro-organisms.


Figure 1: A gel using DNA fluorescence to visualise DNA. The "marker" DNA in the left lane shows the size (larger fragments towards the top of the gel) and quantity of DNA. No DNA is actually visible in the thylacine DNA samples (the four lanes at the right of the gel). © Australian Museum


Figure 2: The same gel as Figure 1 using radioactivity to visualise the DNA. The marker DNA in figure 1 was not made radioactive so is not visible here. The control DNA lanes are given as total amounts of DNA in picograms © Australian Museum

DNA was then extracted from a variety of tissue sources including bone and tooth from two other thylacine specimens (Figure 3) held in the collection of the Australian Museum (a male, specimen M822), and an unsexed individual, specimen M27836). Most of this DNA was less than 500 bases in length (Figure 4). About 2µg of DNA were obtained from the tooth and 1.5 - 2µg from the bone.


Figure 3: Thylacine bone and jaw from which DNA was extracted. Photo: C Bento © Australian Museum


Figure 4: DNA quantitation of tooth and bone DNA using autoradiography. D1-D5 are the control dilution series enabling estimation of DNA amount. © Australian Museum

PCR Amplification

The polymerase chain reaction (which makes many millions of copies of a specified sequence was applied to the DNA samples to confirm the presence of thylacine DNA. "Primer pairs" specifically designed to amplify short fragments (up to 200 base pairs in length) were developed for four DNA sequences for which thylacine sequence information was already available in the GenBank database of the National Center for Biotechnology Information (NCBI) database. (Krajewski et al ., 1992, 1997) .

The primer pairs amplifed two mitochondrial (12S rRNA and Cytochrome b ) and two fragments of a nuclear gene (the protein encoding protamine gene). Samples from the bone and tooth showed very high similarity to the available thylacine DNA sequences for all of these amplifications (Figure 5)


Figure 5: DNA sequencing chromatogram and BLAST results. The DNA sequence is read from left to right (upper panel) by examining which colour (each corresponding to one of the four bases in DNA) predominates at each site. The sequence is then entered to the GenBank database to assess the degree of similarity to other sequences. The 12S gene sequence here is identified as thylacine. © Australian Museum

PCR using primer pairs designed to amplify longer stretches of DNA was not successful on any of the Museum material.

Cloned PCR products

Researchers in the field of ancient DNA are generally concerned by the possibility of PCR artifacts distorting sequences. One method to check for this is to clone the individual DNA molecules produced by PCR reactions to assess the levels of variability between copies of the same gene sequence. The museum has done this for multiple clones from each of the PCR products from the tooth and bone. Details are not presented here as this work forms the substance of a research paper now being written that will shortly be submitted to a scientific journal for publication after peer review.

Attempts at genomic library construction

A number of attempts have been made at construction of genomic libraries from the thylacine DNA. These included standard procedures for using plasmid vectors with DNA made to have sticky ends by A-tailing, and a variety of procedures attempting library construction after whole genomic amplification and/or enzymatic repair of the thylacine DNA. None of these attempts have been successful.

These attempts all were based on the necessity to have inserts of thylacine DNA with as many bases as possible so that once the inserts were sequenced were the pieces could be put into the correct order. With the bone and tooth examples this would have meant inserts of 500 bases. Attempts could have been made to clone much shorter fragments (150 bases, say) but it was decided that this was too inefficient to form a usable genetic library.

The lack of success with attempts to clone longer fragments hugely magnifies the difficulty of making a genetic map of the thylacine for the genomic sequencing planned as the third immediate goal of the project.

References

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