PAK 261

GROUP II
MEMBERS OF GROUP
LONA AMALIA ( 1010413042 )
FRI WARDANA NASUTION ( 1310411087)
ARIF EFENDI (1310411105)

NUCLEIC ACID SEQUENCING
1. Principle of Sanger Sequencing Method
This method begins with the use of special enzymes to synthesize
fragments of DNA that terminate when a selected base appears in the
stretch of DNA being sequenced. These fragments are then sorted
according to size by placing them in a slab of polymeric gel and applying
an electric field a technique called electrophoresis. Because of DNA's
negative charge, the fragments move across the gel toward the positive
electrode. The shorter the fragment, the faster it moves. Typically, each of
the terminating bases within the collection of fragments is tagged with a
radioactive probe for identification.
2. Scheme of Sanger Sequencing Process
a. DNA Sequencing Reaction











b. Gel Electrophoretic Fractionation












3. The Sequencing Result
Quality of sequence data may vary, depending on:
Purity and concentration of template DNA
Presence of extra PCR bands (artifacts)Quality of dye-terminators,
electrophoresis matrix, and other reagents
Ideally, look at chromatograms and convince yourself that base calls are
robust.







4. Illumina Next-Generation Sequencing
With its unprecedented throughput, scalability, and speed, next-generation
sequencing (NGS) enables researchers to study biological systems at a
level never before possible.
Today's complex genomic research questions demand a depth of
information beyond the capacity of traditional DNA sequencing
technologies. Next-generation sequencing has filled that gap and become
an everyday research tool to address these questions.
See What NGS Can Do For You
Next-gen sequencing allows you to:
Rapidly sequence whole genomes
Zoom in to deeply sequence target regions
Utilize RNA sequencing to discover novel RNA variants and splice
sites, or precisely quantify mRNAs for gene expression analysis
Analyze genome-wide methylation or DNA-protein interactions
Use multiplex sequencing with DNA barcode tags to
simultaneously sequence large numbers of samples
Profile microbial diversity in humans or in the environment.


REFERENCES
Applied Biosystems Automated DNA Sequence Chemistry Guide. (2000)

Sanger, F., Nicklen, S., & Coulson, A.R. (1977) DNA Sequencing with chain-
terminating inhibitors. Proc. Natl. Acad. Sci. 94, 5463-5467.

Shendure, J. & Ji, H. (2008) Next-generation DNA Sequencing. Nature
Biotech. 26, 1135-1145

http://thebigone.stanford.edu/papers/Theses/thesis-c.lacenere.pdf
http://www.illumina.com/technology/next-generation-sequencing.ilmn