method (Sanger and Coulson 1975). Restriction fragments from the ϕX174 were
isolated and used as primers in chain elongation using intact ϕX174 as the substrate.
In brief, a primer and target DNAs were placed in four different tubes, the DNA was
denatured, and the primer was allowed to anneal to one of the DNA strands. Each
tube contained regular dNTPs (one labeled with a radioisotope), DNA polymerase,
and a small amount of one of the four dideoxyribonucleotide triphosphates (ddNTPs
– ddATP, ddGTP, ddCTP, and ddTTP). In each reaction tube, the incorporation of a
ddNTP into a DNA molecule resulted in chain termination. The net result was four
tubes each containing a series of fragments of different lengths. The contents of the
tubes were resolved in four adjacent lanes of a polyacrylamide gel. As with MaxamGilbert sequencing, the sequence was deciphered by looking at an autoradiogram of
the gel. Walter Gilbert and Frederick Sanger went on to share a Nobel Prize for their
breakthrough sequencing techniques.
Maxam-Gilbert
6 sequencing was initially the most popular of the two techniques
as it was relatively inexpensive. Moreover, it was strictly chemical in nature and did
not require primers; using primers required some preknowledge of the sequence of
the target DNA or the sequences flanking the target DNA. However, additional
advances in technology would soon see Sanger sequencing gaining a wider following and Maxam-Gilbert sequencing disappear (more or less). With regard to Sanger
sequencing, molecular cloning, ligation of DNA fragments into plasmids of known
sequence, and transformation of bacteria with the recombinant molecules quickly
eliminated the primer problem; one could sequence the insert (or its ends) by using
primers complementary to the plasmid/vector immediately flanking the insertion
site. Moreover, the development of ddNTPs each labeled with a different fluorescent
tag permitted the chain termination reaction to be done in a single tube and
eliminated the dangers of working with radioisotopes. When the polymerase chain
reaction (PCR) emerged on the scene, the amplification of target sequences by PCR
was integrated into the chain elongation/termination step (a process called “cycle
sequencing”). Lastly, use of microcapillary electrophoresis rather than slab gel
electrophoresis improved the throughput of Sanger sequencing and allowed the
technique to be automated. Automated Sanger sequencing became the means by
which the human genome, the Arabidopsis genome, the Caenorhabditis genome,
and all of the initial eukaryotic genomes were sequenced.
The length of reads produced by Sanger and Maxam-Gilbert sequencing techniques was limited by the resolving power of acrylamide gel electrophoresis. While
fragments ranging from 1 to 1,000 nucleotides can be resolved by standard polyacrylamide and/or capillary electrophoresis, polyacrylamide cannot reproducibly
resolve fragments above 1,000 nt that differ by only one nucleotide (Karger and
Guttman 2009).
6 While co-author Peterson has been involved in sequencing many genomes, the only time he
actually did the “hands-on” portion of sequencing himself (i.e., perform reactions, operate apparatuses, etc.) was when he took a short course on Maxam-Gilbert sequencing in graduate school.
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D. G. Peterson and M. Arick
isolated and used as primers in chain elongation using intact ϕX174 as the substrate.
In brief, a primer and target DNAs were placed in four different tubes, the DNA was
denatured, and the primer was allowed to anneal to one of the DNA strands. Each
tube contained regular dNTPs (one labeled with a radioisotope), DNA polymerase,
and a small amount of one of the four dideoxyribonucleotide triphosphates (ddNTPs
– ddATP, ddGTP, ddCTP, and ddTTP). In each reaction tube, the incorporation of a
ddNTP into a DNA molecule resulted in chain termination. The net result was four
tubes each containing a series of fragments of different lengths. The contents of the
tubes were resolved in four adjacent lanes of a polyacrylamide gel. As with MaxamGilbert sequencing, the sequence was deciphered by looking at an autoradiogram of
the gel. Walter Gilbert and Frederick Sanger went on to share a Nobel Prize for their
breakthrough sequencing techniques.
Maxam-Gilbert
6 sequencing was initially the most popular of the two techniques
as it was relatively inexpensive. Moreover, it was strictly chemical in nature and did
not require primers; using primers required some preknowledge of the sequence of
the target DNA or the sequences flanking the target DNA. However, additional
advances in technology would soon see Sanger sequencing gaining a wider following and Maxam-Gilbert sequencing disappear (more or less). With regard to Sanger
sequencing, molecular cloning, ligation of DNA fragments into plasmids of known
sequence, and transformation of bacteria with the recombinant molecules quickly
eliminated the primer problem; one could sequence the insert (or its ends) by using
primers complementary to the plasmid/vector immediately flanking the insertion
site. Moreover, the development of ddNTPs each labeled with a different fluorescent
tag permitted the chain termination reaction to be done in a single tube and
eliminated the dangers of working with radioisotopes. When the polymerase chain
reaction (PCR) emerged on the scene, the amplification of target sequences by PCR
was integrated into the chain elongation/termination step (a process called “cycle
sequencing”). Lastly, use of microcapillary electrophoresis rather than slab gel
electrophoresis improved the throughput of Sanger sequencing and allowed the
technique to be automated. Automated Sanger sequencing became the means by
which the human genome, the Arabidopsis genome, the Caenorhabditis genome,
and all of the initial eukaryotic genomes were sequenced.
The length of reads produced by Sanger and Maxam-Gilbert sequencing techniques was limited by the resolving power of acrylamide gel electrophoresis. While
fragments ranging from 1 to 1,000 nucleotides can be resolved by standard polyacrylamide and/or capillary electrophoresis, polyacrylamide cannot reproducibly
resolve fragments above 1,000 nt that differ by only one nucleotide (Karger and
Guttman 2009).
6 While co-author Peterson has been involved in sequencing many genomes, the only time he
actually did the “hands-on” portion of sequencing himself (i.e., perform reactions, operate apparatuses, etc.) was when he took a short course on Maxam-Gilbert sequencing in graduate school.
128
D. G. Peterson and M. Arick