Biomedical Engineering Reference
In-Depth Information
11.1 DNA Sequencing
The information to produce many of the components of the cell such as RNAs and
proteins is encoded in the sequence of nucleotides of a cell's DNA. Some alterations
to this sequence can contribute to diseases such as cancer, cystic fibrosis, Huntington
disease, to name a few. DNA also contains some of the determinants of an
individual's propensity for some diseases, response to a treatment and prognosis
of clinical course. Determining the DNA sequence is therefore fundamental to
molecular biology and biomedicine, and could potentially open a door to a medicine
tailored for each individual, the ambitious but achievable goal of personalized
medicine. In this section we discuss existing techniques for DNA sequencing.
11.1.1 Sanger's Sequencing Method
The key principle of the Sanger method [
1
,
2
] is the use of dideoxynucleotides
triphosphates (ddNTPs, where the N stands for A, C, G or T) as DNA chain
terminators. As shown in Fig.
11.1a
, during the DNA elongation, the 5
0
-phosphate
group of the dNTPs is condensed with the 3
0
-hydroxyl group at the end of the
extending DNA strand to form a phosphodiester bond by releasing a pyrophos-
phate. If the oxygen atom from the 3
0
-OH group is removed to form a dideoxynu-
cleotide, as shown in Fig.
11.1b
, the DNA strand extension will be terminated
since the phosphodiester bond between two nucleotides cannot be formed under
this condition. The chain terminator dideoxynucleotide triphosphates are used
to convert the DNA sequence information into length information of DNA frag-
ments by polymerase chain reaction (PCR). As shown in Fig.
11.2
, the target DNA
a
b
O
O
5'
O
T
5'
T
O
A
P
CH
2
A
O
O
O
P
O
CH
2
1'
4'
1'
4'
O
O
2'
3'
2'
3'
O
O
O
G
G
O
C
C
O
P
O
CH
2
O
P
O
CH
2
O
O
OH
OH
O
H
O
O
O
C
C
O
G
O
P
O
P
P
CH
2
Dideoxynucleotide
stops synthesis
O
O
O
O
OH
Fig. 11.1 Diagram showing standard DNA synthesis (a) and a chain terminated by the addition of
a dideoxynucleotide chain terminator (b)
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