Page 36 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

330 Chapter 9 Digital Analysis of DNA

template and primer, along with a carefully calibrated The detector transmits information about the signals
mixture of eight nucleotide triphosphates (Fig. 9.7b). to a computer, which shows them as a series of
Four of these are the normal deoxyribonucleotide triphos- different-colored peaks (Fig. 9.7f). The computers in DNA
phates dATP, dCTP, dGTP, and dTTP. The other four are sequencing machines also have base-calling software that
unusual and are added at lower concentrations: They are the interprets the peaks as specific bases and that generates a
dideoxyribonucleotide triphosphates (sometimes just digital file, called a read, of the sequence of As, Cs, Gs,
called dideoxynucleotides) ddATP, ddCTP, ddGTP, and and Ts comprising the newly synthesized DNA. Of course,
ddTTP (Fig. 9.7c). These dideoxynucleotides lack the this sequence is complementary to that of the template
3′ hydroxyl group crucial for the formation of the phos- strand under analysis.
phodiester bonds that extend the chain during DNA DNA sequencing machines available since the late
polymerization (review Fig. 6.21). Moreover, each dide- 1990s can determine about 700–1000 bases from any sin-
oxynucleotide is labeled with a different color fluorescent gle sample. These machines can also run hundreds of
dye; for example, ddATP can carry a dye that fluoresces samples in parallel on separate gel lanes, each recorded
in green, ddCTP has a purple dye, etc.). with a separate fluorescence detector (Fig. 9.7d and e).
The sequencing reaction tube contains billions of orig- Thus, a single machine running for a few hours can deter-
inally identical hybrid DNA molecules in which the oligo- mine hundreds of thousands of bases of DNA sequence
nucleotide primer has hybridized to the template DNA information.
strand at the same location. On each molecule, the primer
supplies a free 3′ end for DNA chain extension by DNA essential concepts
polymerase. The polymerase adds nucleotides to the grow-
ing strand that are complementary to those of the sample’s • In Sanger DNA sequencing, the DNA molecule to be
template strand. The addition of nucleotides continues un- sequenced serves as a template for DNA synthesis by
til, by chance, a dideoxynucleotide is incorporated instead DNA polymerase.
of a normal nucleotide. The absence of a 3′ hydroxyl group • Sanger DNA sequencing requires a short oligonucleotide
in the dideoxynucleotide prevents the DNA polymerase primer that hybridizes to the template. DNA polymerase
from forming a phosphodiester bond with any other nucle- extends the primer by adding (to its 3′ end) nucleotides
otide, ending the polymerization for that new strand of that are complementary to the template.
DNA (Fig. 9.7b). • In automated DNA sequencing, chain synthesis
When the reaction is completed, the newly synthesized terminates when DNA polymerase incorporates a
strands are released from the template strands by denatur- dideoxynucleotide that has a fluorescent label.
ing the DNA at high temperature. The result is a nested set • The DNA fragments made in the polymerization reaction
of fragments that all have the same 5′ end (the 5′ end of the are separated by size on a gel, and a detector reads the
primer) but different 3′ ends. The length and fluorescent color of the fluorescent tag at the 3′ end of each fragment
color of each fragment making up the set is determined to determine the nucleotide sequence.
by the last nucleotide incorporated; that is, the single
chain-terminating dideoxynucleotide the fragment contains
(Fig. 9.7b). 9.4 Sequencing Genomes

The Fluorescence of DNA Fragments learning objectives
Reveals the Nucleotide Sequence 1. Explain why overlap between individual DNA sequences

Biologists analyze the mixture of DNA fragments created is required to reconstruct the sequence of a genome.
by the sequencing reaction through polyacrylamide gel 2. Describe the differences between the hierarchical and
electrophoresis, under conditions that allow the separation shotgun strategies for genome sequencing.
of DNA molecules differing in length by just a single nu-
cleotide (Fig. 9.7b, d, and e). The gel is examined by a
DNA sequencing machine that has a laser to activate the Genomes range from the 700,000 base pairs (700 kb) in
dideoxynucleotide fluorescent tags and a sensitive detector the smallest known microbial genome, to more than 3 billion
that can distinguish the resultant colored fluorescence. As base pairs (3 gigabase pairs, or 3 Gb) distributed among
each DNA fragment passes under the laser, it will glow in the 23 chromosomes of humans, to even larger genomes.
one of the four fluorescent colors dictated by the dye at- Table 9.2 gives the genome sizes of representative
tached to the dideoxynucleotide at the 3′ end of the chain. microbes, plants, and animals. To put these numbers in
Each successive fluorescent signal represents a chain that perspective, the human genome is more than 700 times
is one nucleotide longer than the previous one. larger than that of E. coli and 45 times smaller than the