DNA Analysis Flashcards
(41 cards)
What is the polarity of DNA?
Two opposite strands:
1) 5’ –> 3’
2) 3’ –>5’
Nucleotides are added in a 5’ –> 3’ direction
How is DNA set up?
Nucleotides consist of a deoxy-ribose linked to a nucleotise base
How do the nucleotides in DNA base pair?
A = T, G =/- C
G-C have 3 H bonds
A- T have 2 H bonds
What is molecular hybridization and what does it require?
Hybridization is the annealing of a DNA/RNA strand to its complementary DNA/RNA strand
It requires prior denaturation
It is the underlying principle of many advanced molecular technologies
How does molecular hybridization work?
1) Denature to separate the two DNA strands
2) We design a probe, complementary to the region of interest in the DNA
3) Anneal the probe DNA to hybridize to the target
What is the melting temperature?
The temperature at which half of the DNA with a specific sequence is denatured
C-G has a higher melting temperature due to 3 H bonds, as opposed to two H bonds needing to be broken, leading to more energy needed
Longer strand has a higher melting temperature, as requires more energy to break the bonds
What is the melting temperature (Tm) determined by?
1) Amount of A/T or G/C bonds
2) Clustering of A/T or G/C bonds: Clusters of G/C bonds require high Tm
3) Length of DNA: longer: higher Tm
What is the Tm modulated by?
1) Higher salt increases Tm
2) Denaturing agents decrease Tm
How to do the kinetics of annealing work?
The concentration of complementary DNA strands determine the speed of annealing
Repetitive DNAs will anneal faster than the rare and unique DNAs
Unique DNA is less than 5% in human genomes
More complex DNA: takes longer to anneal
What is the issue with fast annealing?
Fast annealing produces imperfect basepairing, short stretches of single-stranded DNA and other artifacts
How can annealing be slowed down?
Annealing can be slowed down by: High annealing temperature High salt Denaturing agents We use these compounds to reduce imperfect annealing and increase the "stringency" of hybridizations, which is critical for the success of the experiment
What is Southern blotting?
Transfer and immobilize DNA from electrophoresis gel onto a membrane
Detected the presence of specific DNA fragments through molecular hybridization
What is Northern Blotting?
Southern Blotting but for RNA
How is DNA/RNA labeled to be used as a probe?
Without seeing the probe, target DNA/RNA cannot be seen
1) Synthesize In vitro with one of the four nucleotide radioactively or chemically labeled
2) End labeling of DNA
How is the probe labeled using chemical/radioactive labelling?
1) Add 6- mer random oligomer primers
2) Hybridization of these primers to the two denatured DNA strands
3) DNA synthesis with Klenow, polymerase, and four dNTPs, and one of the dNTPs is labeled, say A
4) Denaturation of the probes
There are multiple probe signals that are labeled, so pick up the weak signal, so there is high sensivity, but low specificity because the oligos can bind to other DNA sequences due to the oligos short sequence
How is the probe labeled using end-labelling?
There is a phosphate on the 5’ end of the DNA
1) Add Phosphatase, which removes the phosphate on the 5’ end
2) Add kinase, which is labeled (radioactive), which adds back a radioactive phosphate onto the 5’ end of DNA
Basically exchanging regular phosphate with a radioactive phosphate to detect the probe
How does In Situ Hybridization work (ISH)?
On the spot, which means hybridization on the site of DNA
1) Squash cells on slide
2) Denature DNA (Two single stranded DNAs)
3) Incubate with fluorescent DNA or chemically labeled DNA (probe), then wash to remove unhybridized single strands of DNA
4) Silver grains produced by exposure of emulsion to radioactivity
How does Dual ISH work?
1) Probe co-hybridization (two different probes designed hybridize to two different target sequences)
2) Primary antibody incubation
3) DNA labeled with antibody
4) Detect silver/copy number
What is RNA-ISH?
To analyze the Lebel and cellular localization of specific mRNAs
Same as DNA ISH, except using RNAs
What is a tissue array?
Used to study many tissues in parallel, thousands of small tissue patches are fixed on a glass slide and arranged on an array
How does a tissue array work?
A gene specific probe is used to study transcription levels of the gene in all the tissues on the array simulatiousely for RNA
Tissue arrays can be analyzed using immunohistochemistry ofr immunofluorescence staining with an antibody against a protein, to study protein levels
A gene is expressed in most of the tumours, but not in corresponding healthy tissues
Tissue is placed in the array and ISH can be performed right on the array
How does a DNA microarray work?
1) DNA microarrays are designed with the genes of interest represented on the microarrays
2) DNA (sometimes mRNA) samples are labeled fluorescently and hybridized to the microarray surface
3) After extensive washing, bound DNAs are detected by fluorescence
What is end-point qPCR?
Fluorescence data are collected after the amplification reaction has been completed, usually 30- 40 cycles, and used to back-calculate the amount of template present prior to PCR
Results can be inconsistent
Explain how end-point qPCR works?
There is a Ct value: Signal seen is directly proportional to DNA
A range where DNA is directly proportional to the fluorescence
But, at the end point cannot detect amount of DNA, DNA is not proportional to fluorescence produced
