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DNA sequencing
- Maxan and Gilbert method
- 1 - double stranded DNA is labeled using
radioactive phosphate (phosphate 32) at the 5'
end of each strand
- the attachment of Phosphate 32 is done using a
polynucleotide kinase enzyme and 32P-dATP
- the attachment of Phosphate 32 is done using a
polynucleotide kinase enzyme and 32P-dATP
- 2 - the double stranded DNA is treated with dimethyl
sulphoxide and heated to 90 degree centigrade to
separate the strands
- these strands are then purified
- these strands are then purified
- 3 - the strands are treated with
various chemicals which cause
base modification
- formic acid modifies purines (A and G)
- dimethyl sulphate modifies guanine
- hydrazine modifies pyrimidines
- adding NaCl prevents modification of thymines
- adding NaCl prevents modification of thymines
- formic acid modifies purines (A and G)
- 4 - the altered/modified bases are removed,
hot piperidine is used to cleave the sequence
at modified bases
- the remaining strands of DNA undergo gel electrophoresis, through
these results we can work out what the DNA base sequence is
- the remaining strands of DNA undergo gel electrophoresis, through
these results we can work out what the DNA base sequence is
- 1 - double stranded DNA is labeled using
radioactive phosphate (phosphate 32) at the 5'
end of each strand
- Sanger method
- next generation sequencing
- sequencing by synthesis
- sequencing by ligation
- single molecule sequencing
- sequencing by synthesis
- is finding the order of nucleotides in a piece of DNA
- sequencing can be done on anything
which contains genetic information
- animals
- plants
- bacteria
- archaea
- animals
- depending on the method used you may be provided
with DNA or RNA sequences
- sequencing can be done on anything
which contains genetic information
- sequencing can be used to
determine the sequence of
- individual genes
- large genetic regions such as gene
clusters or operons
- chromosomes
- entire genomes
- individual genes
- the results of sequencing
may be used by
- researchers to further
scientific progress
- by medical proffessionals to aid treatment
decisions and genetic counselling
- researchers to further
scientific progress
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