L4 & 5 Flashcards
When did Sanger publish dideoxy sequencing
In 1977
What does Sanger sequencing do
It relies on DNA polymerase to synthesize a
new strand of DNA, which in turn can reveal the sequence of the target DNA strand.
What does DNA polymerase do
It replicates a new DNA strand complementary to a piece of single stranded DNA
How does DNA pol replicate a new strand
by linking the 5ʹ-hydroxyl end of a free nucleotide to the 3ʹ-OH group of
the nucleotide at the end of a primer.
What is a primer
a small piece of single-stranded DNA that can hybridize to one strand of the
template DNA and be extended by successive additions of nucleotides.
Requirement of Sanger sequencing
a supply of nucleotide triphosphates (dNTPs) and 2ʹ,3ʹdideoxynucleotide triphosphates (ddNTPs) in small quantities relative to dNTPs
ddNTPs contain no reactive 3ʹ-OH hence they are able to?
terminate DNA synthesis once they
are incorporated into the primer extension.
How does DNA synthesis occur
in the 5’ to 3’ direction with the
new nucleotide being added
at the –OH terminus of the
3’carbon
A typical reaction mixture contains
– DNA polymerase.
– DNA to be sequenced.
– DNA primers.
– (dNTPs) dATP, dTTP, dCTP, dGTG.
– Small quantity of one ddNTPs.
– Electrophoresis gel and rig.
When does primer extension stop
until an unmatched nucleotide is paired with a complementary ddNTP.
Many fragments, each ending with a ddNTP of varying length, are produced from such a reaction
What makes fragments detectable by radiography
Radioactive sulphur or phosphorus isotopes are incorporated into the newly synthesized DNA template via labelled dNTPs
What is the goal of the human genome project
to identify all protein coding genes, sequence all chromosomes and make
available via biological publicly available databases.
Key findings of the human genome project include
gene number (~22,000), alternative splicing,
high number of orthologs shared amongst vertebrates, human genome is rich in
transcription factors.
Next Generation Sequencing
refers to novel, commercial technologies that allow us generate millions of sequence
reads in a single sequencing reaction. The length of each sequence read varies from 150-40,000 bp
Roche 454 sequencing
is based on pyrosequencing (requires enzymes), a technique which detects
pyrophosphate release, using light signal (bioluminescence), after nucleotides are incorporated by
polymerase to a new strand of DNA.
Illumina (Solexa) sequencing
works by simultaneously identifying DNA
bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain. Most commonly used
Ion Torrent: Proton / PGM sequencing
g measures the direct release of H+
(protons) from the incorporation of individual bases by DNA polymerase.
Steps of Roche 454 sequencing
1) Library preparation.
2) Clonal amplification.
3) Sequencing.
Library preparation of Roche
DNA is fragmented into single stranded fragments (300-800 bp). Adaptors containing universal priming sites are ligated to target ends. This allows for fragment amplification with common PCR primers.
Clonal amplification of Roche
DNA is mixed in with capture beads (~35µm). These contain adapter primers. Beads are added to PCR reagents and synthetic oil to produce an emulsion.
Steps of Illumina Genome Analyser
1) Library preparation.
2) Clonal clusters amplification.
3) Sequencing.
Library preparation of illumina
DNA fragmented by neubilization or sonication and sequence adapters
are added
Where does cluster amplification occur
On a flow cell
What is a flow cell
A thick glass slide with channels or lanes. Each lane is coated with a lawn of oligos that are complementary to library adapters
Sequencing of illumina
*Flow cell transferred to IGA and sequencing primers, polymerases and
deoxynucleoside triphosphates (dNTP) are added.
Limitation of second generation sequencers
short read lengths (300bp)
What are third generation sequencers
those capable of sequencing single
molecules without the need for DNA amplification
PacBio sequencing or SMRT sequencing
offers much longer read lengths and faster runs than second generation methods but is hindered by a lower throughput, higher
error rate, and higher cost per base.
