Fridge magnate

Professors Robert Lightowlers and Doug Turnbull of the University of Newcastle’s Mitochondrial Research Group owe a special debt to Fred Sanger. Not just because of his two Nobel Prizes, the second of which (for his work on DNA sequencing) laid the foundations for their field. But also for a remarkable feat of memory...

"Can you imagine? After 20 years, finding a test tube the size of the smallest part of your little finger, somewhere in the Sanger labs, which must cover half of Cambridge!" Professor Turnbull laughs: "I suppose it fits: only a sequencer would have the right kind of mind!"

That test tube contained the original DNA sample which, in the late 1970s, Dr Sanger’s group used to sequence the first human genome - the 16 500 base pair human mitochondrial DNA. Now known as the ‘Cambridge Reference Sequence’ (CRS), it has been an indispensable reference for studies of human evolution, population genetics and mitochondrial disease since its publication in 1981.

But what did the Newcastle Group want with the original sample? The story begins in Texas, where Professors Turnbull and Lightowlers, funded by a Biomedical Research Collaboration Grant from the Wellcome Trust, were visiting Professor Neil Howell. The main aim of the study was to combine the Texas group’s fundamental knowledge of mitochondrial genetics with their own more clinically related studies. They found Professor Howell increasingly frustrated by apparent differences between the CRS sequence and what was known from other research.

Mitochondrial DNA (mtDNA) has two unusual characteristics. First, it is extremely variable: mutations are common. And second, it is inherited only through the maternal line: mutations can be clearly followed through generations. They act as ‘markers’ that help to track different human populations and ethnic groups.

But in the CRS some of these common markers ‘didn’t fit’ - in particular, there was a non-European mutation bang in the middle of the reference sequence for European groups. Errors in the original sequencing were one possible explanation, but the differences could simply have reflected individual variation. Not knowing what lay behind the differences was a frustrating stumbling block.

After the visit, Professor Lightowlers decided to phone Professor Alan Coulson in Cambridge with a bizarre request - did any of the original material still exist? Professor Coulson enthusiastically offered to contact Dr Sanger, who had been retired for some time. "And," Professor Lightowlers continues, "He knew where it might be - not only which freezer, but whereabouts in the freezer - and he came in and found it!"

The tiny tube with its precious sample was posted to Newcastle for re-sequencing. In less than three days, they had a sequence that had originally taken the Sanger team more than two years of hard struggle to produce.

What startled them was how accurate the original CRS was. The overall error frequency was a mere 0.05 per cent – Sanger’s team had made just eight sequencing errors in 16 500. "It shows just what a tour de force their work was," comments Professor Turnbull.

Seven other differences were not errors, but rare individual mutations. And three further discrepancies were the result of technical problems Sanger had faced, in the days before automation and new ways of manipulating DNA. Some sections had proved impossible to sequence, and mtDNA from other sources had been used - including DNA of African rather than European origin, which helped resolve the key discrepancy that had bothered Professor Howell so much.

While the work was a sideline to their main research, it has left a personal mark on both men. They had approached Dr Sanger to be a co-author but, says Professor Lightowlers, "In typical Sanger fashion, he wrote back to say he didn’t think he deserved it." Warm admiration is evident in his voice, and the letter is a prized possession.

Professor Turnbull is the same: "Here is one of the great men of science - someone who has made an unbelievable contribution to science - still interested enough to come into the lab and search out a 20-year-old sample for us. It’s a wonderful story."

Further reading

Anderson S, Bankier A T, Barrell B G, de Bruijn M H, Coulson A R, Drouin J, Eperon I C, Nierlich D P, Roe B A, Sanger F, Schreier P H, Smith A J, Staden R, Young I G (1981). Sequence and organization of the human mitochondrial genome. Nature, 290 (5806): 457-65.

Andrews R M, Kubacka I, Chinnery P F, Lightowlers R N, Turnbull D M, Howell N (1999). Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet., 23(2): 147.