1
Q
Detection of DNA in homogenate and in situ?
A
- Homogenate - Southern blotting
- In situ - Chromosomal painting
2
Q
Detection of RNA in homogenate and in situ?
A
- Homogenate - Northern blotting
- In situ - In situ hybridisation
3
Q
Detection of protein in homogenate and in situ?
A
- Homogenate - Western blotting
- In situ - Immunocytochemistry
4
Q
Advantages and disadvantages of detection of DNA, RNA and proteins in the homogenate?
A
Adv. - Quantification - Size - Isolation Disadv. - Require large quantity of tissue for sampling
5
Q
Advantages and disadvantages of detection of DNA, RNA and proteins in situ?
A
Adv. - Tissue distribution of DNA, RNA or protein - Function depending on location Disadv. - Requires tissue processing
6
Q
Steps for detection of DNA, RNA or protein in homogenate?
A
- Separate molecules in gel according to their size
- Separated molecules transferred to membrane and probe is added to detect required molecule
- If molecule is present probe will detect and show up
7
Q
Gel Electrophoresis of DNA
A
- Gel is a porous matrix that acts like a sieve
- DNA has -ve charge so moves toward +ve electrode in gel
- As DNA migrates through gel it separates out according to the size of the molecule - the smaller the molecule the further along it travels
8
Q
4 factors affecting DNA migration in gel electrophoresis?
A
- DNA size - smaller DNA move faster through the gel
- Gel conc. - higher conc. results in slower DNA migration
- DNA shape - supercoiled DNA faster than linear DNA faster than circular DNA
- Gel type - Agarose gels used for DNA fragments of 100-20,000 BPs and polyacrylamide gels used for DNA fragments 10-700 BPs long
9
Q
Agarose or Polyacrylamide gel?
A
- Agarose used for larger range of DNA sizes 100-20,000 BPs
- Polyacrylamide has higher resolution and used for smaller DNA fragments 100-700 BPs long
10
Q
Blotting for DNA and RNA
A
- Relies on the principle of hybridisation
- Hybridisation is the specific base pairing of 2 complimentary single strands to form a double stranded molecule
- Heat is applied to break hydrogen bonds, only the most stable molecules will remain
- Stability of hybridisation depends on the degree of match between target and probe sequence
11
Q
Blotting for Proteins
A
- Relies on the principle of antigen-antibody interaction
- Primary antibody binds to target protein
- Secondary antibody is tagged and binds to primary antibody to allow localisation of target protein
12
Q
In situ hybridisation importance?
A
- Important to use as all tissues have unique subsets of RNA
- Used to detect and quantify mRNA sequences
13
Q
Chromosome painting
A
- Locates specific genes on the chromosome
- Probes labelled with fluorescent colours allows simultaneous viewing of different genes
14
Q
Immunocytochemistry
A
- Relies on the principle of antigen-antibody interaction
- Same method as western blotting
15
Q
DNA Sequencing process?
A
Chain termination method
- DNA to be sequenced used as a template for DNA synthesis in vitro
- DNA to be sequenced needs to be proceeded with some known sequence in order to make a primer to act as a starting point for DNA synthesis
- Terminator nucleotides are added along with normal nucleotides at ratio of 1:100, these prevent subsequent addition of further nucleotides so DNA fragments of different lengths are produced with a known end nucleotide base e.g. Primer+1, Primer+2, Primer+3 etc
- Mixture of DNA molecules passed through polyacrylamide gel electrophoresis
- Terminator nucleotides are tagged with a different colour depending on their base - A, G, T or C
- A detector reads which tagged nucleotide is first to pass through the laser in each fragment
- Used to produce a sequence for unknown DNA strand
16
Q
Restriction enzymes
A
- Precise cutters of DNA - recognise specific DNA sequences and cleave DNA at these sites
- Each enzyme has its own specific restriction site where it will cut
- Isolated from bacteria
17
Q
Restriction sites for enzymes
A
- Short, usually 4-8 BPs
- Sequence is palindromic, the sequence of the sense strand is the same as the antisense strand when rad in the same direction e.g. 5’ to 3’
18
Q
Sticky ends after restriction enzyme cut
A
- Restriction enzymes cut at specific position within its recognition site
- Leaves overhangs in DNA molecules called “sticky ends”
- These can be used to create a new recombinant DNA molecule if the sticky ends of each molecule is complimentary
19
Q
Why are terminator nucleotides added in small numbers during DNA sequencing?
A
- To allow a chance for unknown DNA sequence to be synthesised further down strand
- Too many would produce DNA templates that were too short
20
Q
Polymerase Chain Reaction (PCR)
A
- Used to amplify minute amounts of DNA by repeated cycles of in vitro DNA replication
- DNA amplification proceeds in an exponential scale 2^cycles
21
Q
What do you need for a PCR?
A
- 1 copy of DNA sequence to be amplified - the Template
- Exact DNA sequence at the start and end of DNA sequence to be amplified - used to make Primers
- Primer at the 5’ end derived from sense strand and primer at the 3’ end derived from anti-sense strand
- DNA polymerase and lots of free single nucleotides
22
Q
3 steps in the PCR?
A
- Heat to 95oC for Denaturation of template DNA to separate strands
- Cool to 50-65oC for annealing of primers to template DNA
- Heat to 72oC for elongation of primers till end of template
Cycle starts again with products then acting as templates
23
Q
How can PCR be used to manipulate DNA sequences?
A
- Primers can be used to insert single point mutations by lowering temperature for annealing to allow imperfect binding between primer and DNA sequence
- Tailed primers can be used to insert restriction enzyme sites to produce ‘sticky ends’ for DNA engineering
24
Q
DNA Cloning
A
- Insert DNA (with sticky ends from restriction enzymes) into a vector such as a plasmid
- Use DNA ligase to fuse the DNA and plasmid
- Introduce recombinant DNA into the bacteria
- Use antibiotic selection for bacteria containing plasmids and this DNA is purified
25
Vector
A DNA molecule that is maintained and replicated naturally by a host organism e.g. a plasmid
26
3 essential properties of cloning plasmid?
- Contain an origin of replication to allow it to replicate independently of the bacterial chromosome
- Contain anti-biotic resistance genes to select for bacteria containing recombined DNA
- Restriction enzyme sites to allow insertion of insert DNA
27
Expression plasmids must contain what to initiate transcription of the insert?
Promotor region
28
Genomic libraries
Population of identical vectors containing different inserts and used to clone all DNA sequences from a cell
29
cDNA libraries
- Population of identical vectors containing different inserts and is derived from mRNA thus represents the part of the genome that is made into mRNA
- Can be derived from different organs or stages of development
30
Microarrays
- Performed in a cell free system using isolated mRNA
- Monitors expression of 1000s of genes at once
- Compares transcribed genes in 2 tissues or conditions of same tissue
31
siRNA for Gene Knockdown
- Small interfering RNA
- Interfere with expression of genes causing a decrease in expression of the target gene
- Uses RNA interference pathway in cells, which regulates gene expression
32
2 methods to generate transgenic mice?
- Pronuclear injection
| - Gene targeting
33
Pronuclear injection to generate transgenic mice
- A foreign gene is inserted into the nucleus of a fertilised ova
- Several copies are inserted at random sites in the genome
- Used to generate GM crops and animals
34
Gene targeting to generate transgenic mice
- Foreign DNA is introduced into cultured mouse stem cells
- Foreign DNA integrates at specific sites in genome
- Modified ES cells transferred to blastocyst and inserted into foster mother which give birth to chimeric mice
- Chimeric mice bred with normal mice to produce some gene targeted offspring
- This is used to generate insertions (knockins) and deletions (knockouts)
35
Yeast 2 Hybrid Screen
- Screens for interacting proteins, based on transcriptional activation
- Protein of interest (bait) is bound to DNA binding domain and proteins that bind to bait (fish) are bound to activation domains
- Any protein that binds to bait will activate expression of the reporter gene
- Construct a bait plasmid and library of cDNA fish plasmids, with each type of plasmid containing a marker such as an essential amino acid
- Plasmids transformed into yeast cells, which are placed in a medium lacking the essential aa marker, so only those containing both plasmids will grow
- Remaining cells transferred to agar plate lacking product of reporter gene to isolate cells containing interacting genes
- Binding proteins identified by sequencing DNAs of plasmids isolated from these cells
36
REFLP
- Restriction Enzyme Fragment Length Polymorphism
- Point mutations can lead to abolition or addition of restriction enzyme sites
- So a mutation may create a polymorphism in the number/sizes of DNA fragments produced by a particular restriction enzyme.
- This change becomes a marker for the mutation
37
DNA Fingerprinting
- Based on profiling specific regions in our genome
- These regions contain repeats of certain short sequences
- The number of repeats within each region varies between individuals (Variable Number of Tandem Repeats)
- Location of repeats does not change between individuals
BMS 109
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