Download Southern hybridization

DNA SEQUENCING
Principles & methods
Topic Outline
Principles of DNA Sequencing
Methods of DNA Sequencing
Principles of DNA Sequencing
Denaturation
Cycle
sequencing
(one cycle)
Extension/
termination
Annealing
 Partial copies of DNA
fragments made with
DNA polymerase
 Collection of DNA
fragments that terminate
with A,C,G or T using
ddNTP or chemical.
 Separate by gel
electrophoresis
 Read DNA sequence
Radioactive label
Denaturation, Gel
electrophoresis
Four aliquots – treated with
chemicals ( modification)
Cleaved by piperidine
Separated by Electrophoresis
Synthesis
termination
Dead-end
product
Add in DNA
polymerase I,
dNTPs, ddNTPs
Extension /synthesis
of complementary
strands
Run gel divide by
size
Running the reaction of all
the dideoxy nucleotides
using different dyes
generates this type of
diagram in same lane.
 Sometimes the spacing
between the bands is hard to
measure.
 Thus use machine to run and
read the electrophoresis.
 Capillary electrophoresis: the
fragments are piped through a
tiny glass-fiber capillary
during the electrophoresis
step, and they come out the far
end in size-order.
 Chemical cleave method
 Sequence small fragments
of DNA
 The radioactive labelling is
done on the dsDNA.
 Division of aliquots is done
by methylation or removal
of base.
 Requires DNA
 Breaks DNA at different
nucleotides
 Enzymatic cleave method
 Sequencing small fragments are
problematic.
 The radioactive labelling is done
on the ssDNA.
 Allow high throughput
automated sequencing
techniques.
 Allow Real Time detection.
 Requires DNA synthesis
 Termination of chain elongation
SOUTHERN, NORTHERN AND
WESTERN BLOTTING
Comparison of Southern, Northern,
and Western analyses of Gene X
SOUTHERN BLOTTING
 The technique was developed by E.M.
Southern in 1975.
 The Southern blot is used to detect the
presence of a particular piece of DNA in a
sample.
 The DNA detected can be a single gene, or it
can be part of a larger piece of DNA such as a
viral genome.
Southern hybridization
Transfer buffer
Detection of an RFLP by
Southern blotting
Flow chart of Southern
Preparing the samples and running the gel
hybridization
Southern transfer
Probe preparation
Isotope
Non-isotope
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
SOUTHERN BLOTTING
 The key to this method is hybridization.
 Hybridization-process of forming a double-
stranded DNA molecule between a singlestranded DNA probe and a single-stranded
target patient DNA.
SOUTHERN BLOTTING
 There are 2 important features of
hybridization:
 The reactions are specific-the probes will only
bind to targets with a complementary sequence.
 The probe can find one molecule of target in a
mixture of millions of related but noncomplementary molecules.
Comparison of Southern, Northern,
and Western blotting techniques
Molecule
detected
Gel
electrophoresis
Gel
pretreatment
Southern blotting
DNA (ds)
Northern blotting
mRNA (ss)
Western blotting
Protein
Agarose gel
Formaldehyde
agarose gel
-
Polyacrylamide gel
Capillary transfer
cDNA, cRNA
Radioactive or
nonradioactive
Autoradiography
Chemiluminescent
Colorimetric
Electric transfer
primary antibody
Depurination,
denaturation, and
neutralization
Blotting method Capillary transfer
Probes
DNA
Radioactive or
nonradioactive
Detection
Autoradiography
system
Chemiluminescent
Colorimetric
-
Chemiluminescent
Colorimetric
MB206
MUTAGENESIS
What Is a Mutation?

Genetic information is
encoded by the sequence of
the nucleotide bases in DNA
of the gene. The four
nucleotides are: adenine (A),
thymine (T), guanine (G), and
cytosine (C), a mutation is a
change in the order of these
nucleotides.

A change in the order can cause
the gene to encode for wrong
proteins and inhibit the function
of the gene or cause the gene to
be virtually inactive.
Mutagenesis
 Mutagenesis (the creation or formation of a
mutation) can be used as a powerful genetic
tool. By inducing mutations in specific ways
and then observing the phenotype of the
organism the function of genes and even
individual nucleotides can be determined.
Some types of Mutagenesis
 Directed Mutagenesis
 Site-directed/Site-specific Mutagenesis
 Mismatched Mutagenesis