Genetic Engineering Flashcards
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
Q
Vector
A
A DNA molecule that is maintained and replicated naturally by a host organism e.g. a plasmid
26
Q
3 essential properties of cloning plasmid?
A
- 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
Q
Expression plasmids must contain what to initiate transcription of the insert?
A
Promotor region
28
Q
Genomic libraries
A
Population of identical vectors containing different inserts and used to clone all DNA sequences from a cell
29
Q
cDNA libraries
A
- 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
Q
Microarrays
A
- 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
Q
siRNA for Gene Knockdown
A
- 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
Q
2 methods to generate transgenic mice?
A
- Pronuclear injection
- Gene targeting
33
Q
Pronuclear injection to generate transgenic mice
A
- 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
Q
Gene targeting to generate transgenic mice
A
- 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
Q
Yeast 2 Hybrid Screen
A
- 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
Q
REFLP
A
- 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
Q
DNA Fingerprinting
A
- 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
