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HYBRIDISATION TECHNIQUES
SOUTHERN BLOTTING
Definition
-Method used in Molecular Biology for detection of specific DNA sequences in DNA samples
...
Southern (1975)
-Hybridisation technique for identification of particular size of DNA from the mixture of other similar
molecules
-Combines transfer of electrophoresis-separated DNA fragments to a filter membrane and
subsequent fragment detection by probe hybridisation
...

-DNA fragment on basis of size and charge during electrophoresis
-Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using
specific DNA probe that is complementary to the desired DNA
-Hybridisation probe is a short single-stranded DNA
-Probes labelled with a marker so that they can be detected after hybridisation

Procedure
I
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III
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V
...

VII
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STEP 7


Membrane bound DNA labelled with probe can be visualised under autoradiogram which
gives pattern of bands

Applications
a) Detect DNA in a given sample

b)
c)
d)
e)
f)
g)
h)

DNA fingerprinting
Paternity testing, criminal identification, victim identification
Isolate and identify desired gene of interest
Used in restriction fragment length polymorphism
Identify mutation/gene rearrangement in the sequence of DNA
Identify infectious agents
Diagnosis of disease caused by genetic defects

NORTHERN BLOTTING
Definition
Technique used to study gene expression by detection of RNA in a sample
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II
...

IV
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VI
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Separated RNA sequence transferred to nylon membrane
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Fixed nylon membrane is then mixed with probes
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Labelled probe detected by chemiluminescence/autoradiography
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Principle
-Rapid & sensitive assay for detection and characterisation of proteins
-Based on the principle of immune-chromatography where proteins are separated into
polyacrylamide gel according to their molecular weight

-Proteins thus separated are transferred/electrotransferred onto nitrocellulose membrane
-Detected using specific primary antibody and secondary enzyme labelled antibody and substrate

Procedure
A
...

-Lysis buffer should contain protease inhibitors to prevent degradation of protein of interest
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-Cell mixture centrifuged and pellet discarded
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Gel Electrophoresis
-Prepare the gel by inserting in the electrophoresis apparatus
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C
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-Notch the top left corner to indicate gel orientation
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-Notch the top left corner of blotting paper to indicate blot orientation
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-Sponge, filter paper, gel, membrane, filter paper and sponge
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Immuno blotting
-Remove membrane and rinse 3 times
-Block areas of blot not containing protein using blocking buffer BSA in a TBS-Tween
solution
...
t
-Wash with TBS tween for 5 mins
...

-After washing dilute 20 antibody in the blocking solution and incubate for 1 hour at rt
-Decant membrane and wash with large volumes of TBS
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Detection
-Radioactive labels on the blot
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-Creates dark regions
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Principle
-Developed by Kary Mullis in 1983
-5 core materials
DNA template to be copied
Primers, short stretches of DNA that initiate PCR reaction
DNA nucleotide bases
Taq polymerase
Buffer
-Involves a process of heating and cooling called thermal cycling
 3 main stages
Denaturation
When ds template DNA is heated to separate it into 2 single strands
Annealing
When the temperature is lowered to enable DNA primers to attach to the template
Extension
When temperature is raised and the new strand of DNA is made by the Taq
polymerase enzyme
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of DNA each time
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Procedure
 Denaturation stage
-Mixture containing the template DNA and all other reagents is heated to 94-950C
...

-Results in 2 single strand of DNA which act as templates for production of new strands of
DNA
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-Usually takes 15 – 30s

 Annealing Stage
-Reaction cooled to 50 – 600C
-Enables primers to attach to a specific location on the ss template DNA by way of H-bonding
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Designed to be complementary in sequence to short sections of DNA

Serves as starting point for DNA synthesis

Polymerase can only add DNA bases to a dsDNA

-Once the primer has bound, the polymerase can attach and start making new complementary
strand of DNA
...

 Extension Stage
- Heat increased to 720C to enable new DNA to be made by a special Taq DNA polymerase
which adds DNA bases
...

-Attaches to primer and adds DNA bases to the ss one-by-one in 5’ – 3’ direction
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-Usually takes around 1 min
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II
...


IV
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Real-time PCR
-Used to reverse-transcribe and amplify RNA to cDNA
-PCR preceded by a reaction using reverse transcriptase – converts RNA to cDNA
Uses
-Detection of RNA virus
-Detection of other microorganisms through targeting of their rRNA
Nested PCR
-Used to increase the specificity of DNA amplification
-2 sets of primers used in 2 successive reactions
-First – one pair of primers used to generate DNA products which will be the target for the
second reaction
-Using one /two different primers whose binding sites located within the first set, thus
increasing specificity
Uses
-Detection of pathogens that occur with very few amount
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Applications
-Identification and characterisation of infectious agents
-Genetic fingerprinting
-Detection of mutation
-PCR sequencing
-Cloning genes

SEQUENCING – DIDEOXY CHAIN TERMINATION
METHOD
(SANGER SEQUENCING)
Definition
DNA sequencing is the process of determining the sequence of nucleotides (A, T, C, G) in a piece of
DNA
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Principle
-A DNA primer is attached by hybridisation to the template strand and deoxynucleotides
triphosphates (dNTPPs) are sequentially added to the primer strand by DNA polymerase
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-M13 sequences are generally attached to 3’end & the primer of this M13 is made
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-If during replication, ddNTPs is incorporated instead of usual dNTPs in the growing DNA strand then
the replication stops at that nucleotide
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-DNA primer is essential to initiate replication of template
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 Generation of nested set of labelled fragments
-Copies of each template divided in 4 batches & each batch is used for different replication
reaction
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-To synthesise fragments that terminates at A, ddATP is added to the reaction mixture on
batch I along with dATP, dTTP, dCTP & dGTP, standard primer & DNA Pol I
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Electrophoresis & Gel reading
-Reaction mixture from 4 batches loaded into 4 different wells on polyacrylamide get &
electrophoresed
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-Band of shortest fragments are at the bottom of autoradiogram so that the sequences of C
strand is read from bottom to top

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