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BMS 109 > Genetic Engineering > Flashcards

Genetic Engineering Flashcards

1
Q

Detection of DNA in homogenate and in situ?

A
  • Homogenate - Southern blotting

- In situ - Chromosomal painting

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2
Q

Detection of RNA in homogenate and in situ?

A
  • Homogenate - Northern blotting

- In situ - In situ hybridisation

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3
Q

Detection of protein in homogenate and in situ?

A
  • Homogenate - Western blotting

- In situ - Immunocytochemistry

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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
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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
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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
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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
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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
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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

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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
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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
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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

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13
Q

Chromosome painting

A
  • Locates specific genes on the chromosome

- Probes labelled with fluorescent colours allows simultaneous viewing of different genes

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14
Q

Immunocytochemistry

A
  • Relies on the principle of antigen-antibody interaction

- Same method as western blotting

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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
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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
  1. Heat to 95oC for Denaturation of template DNA to separate strands
  2. Cool to 50-65oC for annealing of primers to template DNA
  3. 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

BMS 109 (8 decks)

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