individual restriction fragments from mixtures. And most impor-
tantly, to do DNA sequencing, at least three steps are needed:
cloning, sequencing, and analyzing.
In the year 1970, Ray Wuat from Cornell University first
tapped a location-specific primer extension strategy into determin-
ing DNA sequences. And ever since the year 1977 Sanger and his
colleagues established the chain termination method and accom-
plish the first full DNA genome sequencing of bacteriophage
φX174, DNA sequencing methods have been developed and
improved.1.1.1 Traditional
Methods
There are two basic methods in DNA sequencing—the Maxam-
Gilbert sequencing (also known as chemical sequencing) as well as
the chain termination method (also known as Sanger sequencing).
The former method attaches radioactive labels to the 5^0 end of
DNA, and by using chemical treatment, it generates subsequent
breaks at particular bases. Autoradiography helps yield a series of
dark bands, which represent the radiolabeled DNA fragments. On
the other hand, Sanger’s method requires modified
di-deoxynucleoside triphosphates (ddNTPs). Due to the fact that
DNA polymerase I cannot distinguish normal deoxynucleoside
triphosphates (dNTPs) and ddNTPs, those new strands with
ddNTPs lack a 3^0 -OH group required for the formation of a
phosphodiester bond between two nucleotides, thus stopping the
elongation of DNA. By labeling ddNTPs, we get to know the DNA
sequence [2].
Although Sanger’s way is effective in many aspects, it can only
read 450 bp in a single reaction, and the process is time-consuming,
limiting its use in large fragment sequencing. After prevailing for
decades, other methods are invented and widely used on the basis
of their work, like the shotgun strategy and bridge PCR. More
importantly, with the rapid development of science and technology,
high-throughput sequencing methods are established; they then
play an essential role in modern DNA sequencing with the ability to
process mass data in a short time.1.1.2 High-Throughput
(HTP) Sequencing Methods
Since the 1990s, a handful of new methods of DNA sequencing
were invented—454 pyrosequencing, Illumina (Solexa) sequenc-
ing, and SOLiD sequencing are three most used technologies.
Other methods include the massively parallel signature sequencing
(MPSS), the polony sequencing, DNA nanoball sequencing, etc.
These methods all share common characteristics of high through-
put and low costs, and together they were known as the “next-
generation” sequencing (NGS) methods. The core thought of
HTP methods is to do DNA sequencing while synthesizing the
new strand.2 Keyi Long et al.