21-06-2014, 04:04 PM
DNA sequencing by the Sanger method
[attachment=65174]
DNA sequencing example
Problem Statement: Consider the following DNA
sequence (from firefly luciferase). Draw the sequencing
gel pattern that forms as a result of sequencing the
following template DNA with ddNTP as the capper.
Reading a sequencing gel
You begin at the right, which are the smallest DNA fragments.
The sequence that you read will be in the 5'-3' direction.
This sequence will be exactly the same as the RNA that
would be generated to encode a protein. The difference is that
the T bases in DNA will be replaced by U residues. As an example,
in the problem given, the smallest DNA fragment on the sequencing
gel is in the C lane, so the first base is a C. The next largest band
is in the G lane, so the DNA fragment of length 2 ends in G.
Therefore the sequence of the first two bases is CG.
The sequence of the first 30 or so bases of the DNA are:
CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC..
When this is made as RNA, its sequence would be:
CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC..
Note that the information content is the same, only the T's have
been replaced by U's!.
Translating the DNA sequence
The order of amino acids in any protein is specificed by the
order of nucleotide bases in the DNA.
Each amino acid is coded by the particular sequence of three bases.
To convert a DNA sequence
First, find the starting codon. The starting codon is always
the codon for the amino acid methionine. This codon is
AUG in the RNA (or ATG in the DNA):
GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC..
Met
In this particular example the next codon is AAG. The first base
(5'end) is A, so that selects the 3rd major row of the table. The
second base (middle base) is A, so that selects the 3rd column of
the table. The last base of the codon is G, selecting the last line in
the block of four.
High-throughput seqeuncing: Capillary electrophoresis
The human genome project
has spurred an effort to
develop faster, higher
throughput, and less
expensive technologies
for DNA sequencing.
Capillary electrophoresis
(CE) separation has many
advantages over slab gel
separations. CE separations are faster and are capable of producing
greater resolution. CE instruments can use tens and even
hundreds of capillaries simultaneously. The figure show a simple
CE setup where the fluorescently-labeled DNA is detected as it
exits the capillary.
[attachment=65174]
DNA sequencing example
Problem Statement: Consider the following DNA
sequence (from firefly luciferase). Draw the sequencing
gel pattern that forms as a result of sequencing the
following template DNA with ddNTP as the capper.
Reading a sequencing gel
You begin at the right, which are the smallest DNA fragments.
The sequence that you read will be in the 5'-3' direction.
This sequence will be exactly the same as the RNA that
would be generated to encode a protein. The difference is that
the T bases in DNA will be replaced by U residues. As an example,
in the problem given, the smallest DNA fragment on the sequencing
gel is in the C lane, so the first base is a C. The next largest band
is in the G lane, so the DNA fragment of length 2 ends in G.
Therefore the sequence of the first two bases is CG.
The sequence of the first 30 or so bases of the DNA are:
CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC..
When this is made as RNA, its sequence would be:
CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC..
Note that the information content is the same, only the T's have
been replaced by U's!.
Translating the DNA sequence
The order of amino acids in any protein is specificed by the
order of nucleotide bases in the DNA.
Each amino acid is coded by the particular sequence of three bases.
To convert a DNA sequence
First, find the starting codon. The starting codon is always
the codon for the amino acid methionine. This codon is
AUG in the RNA (or ATG in the DNA):
GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC..
Met
In this particular example the next codon is AAG. The first base
(5'end) is A, so that selects the 3rd major row of the table. The
second base (middle base) is A, so that selects the 3rd column of
the table. The last base of the codon is G, selecting the last line in
the block of four.
High-throughput seqeuncing: Capillary electrophoresis
The human genome project
has spurred an effort to
develop faster, higher
throughput, and less
expensive technologies
for DNA sequencing.
Capillary electrophoresis
(CE) separation has many
advantages over slab gel
separations. CE separations are faster and are capable of producing
greater resolution. CE instruments can use tens and even
hundreds of capillaries simultaneously. The figure show a simple
CE setup where the fluorescently-labeled DNA is detected as it
exits the capillary.