Page 33 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

9.3 Sequencing DNA 327

9.3 Sequencing DNA clones in the library) but not of the genomic DNA insert
(which will vary in different clones) (Fig. 9.7a).
Next, you would need a short oligonucleotide primer
learning objectives complementary to the known sequence of the vector just
adjacent to the unknown human DNA insert (Fig. 9.7a).
1. Explain the roles of DNA polymerase, the template, and Primers are made to order in DNA synthesizers, machines
the primer in a Sanger sequencing reaction. that can manufacture large quantities of any given DNA
2. Describe the role of dideoxyribonucleotides in oligonucleotide up to 100 bases in length. The user simply
generating DNA fragments for analysis. types in the desired sequence of nucleotides into the com-
3. Interpret the fluorescent peaks obtained during a DNA puter controlling the DNA synthesizer, and the machine
sequencing run as a sequence of nucleotides with the then strings those nucleotides together in the proper order
proper polarity. using chemical reactions. You can design the primer be-
cause you already know the sequence of the vector, which
was determined by alternative chemical techniques (not de-
Looking at Fig. 9.6, you see petri plates containing thou- scribed here) that do not require prior knowledge.
sands of separate colonies constituting part of a human ge- Sanger sequencing allows the template and primer de-
nomic library. Each clone contains a different recombinant picted in Fig. 9.7a to interact through the process of
DNA molecule, each with a plasmid vector attached to a hybridization: the natural tendency of complementary
different fragment of human genomic DNA. Note that the single-stranded molecules of DNA or RNA to base pair and
colonies are scattered randomly around the plate, so their form double helixes. To make the template, you could sim-
arrangement on the plate has no correspondence to their ply amplify and purify double-stranded recombinant DNA
relative order in the genome. How can you then tell which from one particular clone, and then melt the DNA into sin-
colony contains which fragment of human DNA? gle strands by raising the temperature so as to disrupt the
With the technology available today, the simplest way hydrogen bonds that would otherwise keep the strands to-
to answer this question is to sequence the human DNA in- gether. Although both strands of a DNA fragment are pres-
sert in each clone. The DNA sequencing technology in ent in a typical DNA sample, only one is used as a template
widest current use is based on an original method devel- for sequencing. You then mix in large amounts of the pre-
oped in the mid-1970s by Fred Sanger, who won one of his viously made primer. As the temperature of the mixture is
two Nobel Prizes for this work (the other was awarded for gradually lowered, hydrogen bonds will form between
his determination of the amino acid sequence of the hor- complementary nucleotides of the primer and the template
mone insulin). Sanger’s methodology can be automated strand of the recombinant DNA. The primer you make in
easily, providing the power needed to sequence the 3 billion the DNA synthesizer must be long enough to ensure that it
nucleotides in the human genome. will form a stable double-stranded region (that is, anneal)
only with the one complementary sequence in the template;
usually primers are between 17 and 25 bases long. The in-
Sanger Sequencing Depends teraction of primer and template creates the substrate for
on DNA Polymerase the action of DNA polymerase (Fig. 9.7a).

Sanger based his technique on his knowledge of the way DNA
is replicated in cells. You will recall from Chapter 6 that the Sanger Sequencing Generates Nested
enzyme DNA polymerase catalyzes DNA replication. As Sets of Single-Stranded DNA Fragments
summarized in Fig. 9.7a, this enzyme’s minimal requirements
are: (1) a template; that is, a single strand of DNA to copy; To reveal the order of base pairs in an isolated DNA mol-
(2) deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, ecule, Sanger sequencing uses DNA polymerase to create a
and dTTP) that are the basic building blocks for newly synthe- series of single-stranded fragments, where part of each frag-
sized DNA; and (3) a primer; that is, a short single-stranded ment is complementary to the unknown portion of the DNA
DNA molecule (an oligonucleotide) that is complementary to template under analysis (Fig. 9.7b). Each fragment differs
part of the template and that provides the free 3′ end to which in length by a single nucleotide from the preceding and suc-
DNA polymerase can attach new nucleotides. ceeding fragments; the graduated set of fragments is known
To sequence DNA using Sanger’s methods, you would as a nested array. A critical feature of the fragments is that
need a template, part of whose sequence is known, but the each one is distinguishable according to its terminal 3′ base.
remainder of which is unknown (because that is what you are Thus, each fragment has two defining attributes—relative
trying to determine). One strand of the DNA of a recombi- length and one of four possible terminating nucleotides.
nant plasmid could serve as such a template: You know the The sequencing procedure to create the nested array
DNA sequence of the vector (which will be the same in all begins by adding DNA polymerase to the annealed