F.
Sanger, S. Nicklen and A.R. Chase. 1977.
DNA
Sequencing with Chain Terminating Inhibitors. Proc. Natl. Acad. Sci.
USA.
74(12): 5463 – 5468.
Presented by: Caitlin Hill, Becky Richardson and Michelle Wille
Outline:
Biography
DNA Sequencing: How did we get here?
Steps towards DNA Sequencing
The concept
How it all works?
Critique
Why should we care? Impacts of the experiment, and evolution of DNA Sequencing.
Fred Sanger Biography
DNA
Sequencing: How did we get here?
Throughout history, numerous individuals have contributed to the study of genetics and biochemistry, and advanced our knowledge of nucleic acids so that we have contrived the concept of sequencing that which we cannot see, but believe exists.
1859: Charles
Darwin publishes the Origin of Species which describes his theory
of
natural selection, which states that members of a population who are
better
adapted to the environment are more likely to survive and pass on their
traits.
1866: Gregor Mendel publishes his results of the pea experiments, through which he created the laws of inheritance
1882: Walter Flemming discovers rod shaped bodies through staining which he names “chromosomes”
1906: The term “genetics” is used for the first time.
1909: Wilhelm Johannsen proposes the terms “gene”, “genotype” and “phenotype”.
1915: Thomas Hunt Morgan publishes “The Mechanism of Mendelian Heredity” presenting his results from experiments of fruit flies. He describes the principals of linkage and alleles. A “gene map” is created to illustrate the locations of genes responsible for numerous traits – the principals of gene mapping.
1952: Alfred Hershey and Martha Chase provide evidence through phages, that the genetic material is stored in nucleic acids, not amino acid based proteins.
1953: Francis
Crick and James Watson determine the structure of ΦX174 , and create
the
double helix model.
1958: Matthew Meselson and Franklin Stahl provide evidence to support the semi-conservative model for ΦX174 replication.
1961: Sydney Brenner, Francois Jacob and Matthew Meselson identify the role of RNA, and determine that mRNA is the molecule that carries the genetic information from DNA to make proteins.
1961: Francois Jacob and Jaques Monod describe genetic regulatory mechanisms using the lac operon as a model.
1966: Marshall Nirenberg and H. Gobind Khorana crack the genetic code – each of the 20 amino acids is coded by a sequence of three nucleotide bases and termed the word “codon”
1977: Frederick Sanger develops the chain termination method to sequence ΦX174 .
Steps
towards DNA Sequencing
This was not the first method developed to sequence DNA, numerous other methods existed and were considered to create the chain-terminating inhibitor method.
The Plus and Minus Method (1975) was one of the first methods used to sequence DNA, and required a comparison of both the “plus” and “minus” sequences to determine the actual sequence. It could be used only for sequencing ssDNA
The Maxim and Gilbert Method (1977) was published just before the inhibitor method, however it required a series of complex reactions, and was therefore a much more difficult method of sequencing. Like the inhibitor method, it can be applied to dsDNA.
The Sanger
et al (1977) method of DNA Sequencing
was a method that incorporated the idea that inhibitors can terminate
elongation of DNA at specific points.
How
it all works: Methods and Results
In 1977 dideoxynucleotides were not commercially available. Not only were they not commercially available, but ddCTP and ddGTP did not exist.
The first step was the preparation of triphosphate analogues.
Complementary ΦX174,
restriction
enzyme fragments from replicative ΦX174 and
buffer were combined and incubated before being diluted. Each sample
was then
mixed with DNA polymerase, dATP and the
appropriate dNTP and ddNTP before being
incubated again. There were four samples, each with one of the ddNTPs. After the final incubation, more dATP was added as a “chase” step to prevent
termination at A residues.
Each sample was then denatured
and applied
to acrylamide gel next to one another for
electrophoresis.
Sequencing Animation:
How to read an
audioradiograph
Figure 1: DNA Sequence of ΦX174 using short primers
Figure 2: DNA Sequence of ΦX174 DNA using a longer primer
Figure 3: DNA Sequence
of ΦX174 DNA using the single-site ribosubstitution
method
Autoradiogram from Carr lab
Method
critique:
There are numerous advantages to this method in comparison to the “plus and minus” method sequencing used prior to 1977.
1.) Simple – no preliminary extension is required, therefore avoiding incubation and purification.
2.) It requires commercially available DNA Polymerase I.
3.) The results are better with fewer artifacts and a larger sequence can be read
4.) Intermediate nucleotides in
“runs” show
up in bands, which avoids estimating the number of nucleotides in each
runs.
The first nucleotide of the run is usual the strongest
(except in ddCTP), and in long runs one
can estimate the number of
nucleotides by the strength and width of each band.
5.) This method works on a small scale as well as a large one, due to better incorporation of 32P and longer incubation period resulting in longer extension
6.) 15-200 nucleotides can be read from primer site, and can be determined with accuracy. Up to 300 nucleotides is possible
Some disadvantages of the method
1.) There are occasional artifacts, likely due to contaminant fragments
2.) Band “pile-ups” can occur
due to loop
formation under gel conditions, and is usually depicted as numerous
bands in
the same position, or very close together on the gel.
These pile-ups
appear at
different positions when the priming is done in opposite directions.
This can be seen in Figure 2 (4300 – we see 1 band where there should be 3C’s due to pairing with 4C’s at 4323) and Figure 3 (3545-3550)
3.) Though the method is more reliable than the “plus and minus” method, is is not completely accurate and therefore repeating the sequencing more than once, or comparing the result is important.
4.) In 1977 it was still difficult to obtain all the inhibitors, particularly ddGTP, and therefore these had to be prepared in advance from other molecules.
Why should
we care? Impacts of the experiment, and evolution
of DNA
Sequencing.
1983 - The gene for a human genetic disease is mapped to a specific human chromosome.
1984 - Alec Jeffreys introduces DNA fingerprinting as a method of identification.
1990 - The Human Genome Project is launched.
1992 - Daniel Cohen produces a map that includes genetic markers on all 23 human chromosomes.
1994 - A high density genetic map of the human genome, consisting of almost 6,000 markers, is published in Science magazine.
1995 - The first full genome sequence of a living organism other than a virus is completed for the bacterium Hemopilus influenzae by Craig Venter.
1997 - Dolly
1998 - A human genetic map is produced, showing the chromosomal locations of markers from more than 30,000 human genes.
2000 - A rough draft of the human genome is published by the Human Genome Project and Celera.
2003 - 50th anniversary of the discovery of the double-helix structure of DNA by Watson and Crick.
Automated Sequencing, a more accurate and efficient means of sequencing double-stranded DNA, is a widely used method today.