8. RECOMBINANT DNA & CLONING Flashcards
1
Q
What are the 4 main recombinant vectors used?
A
- Plasmids
- Viruses
- Bacteriophages
- Artificial Chromosomes
2
Q
What are plasmids?
A
- Plasmids are discrete, circular double stranded molecules found in many but not all bacteria
- Plasmids can exist & replicate independently of the chromosome so they are extra-chromosomal
- Plasmids have a restricted host range
- Plasmids are a way of storing genetic material, it can be transferred via transformation or conjugation (sex pilli)
3
Q
***What are bacteriophages?
A
- Bacteriophages are viruses that infect bacteria, they are known as Lamda
- Viruses can transfer their genetic material into bacteria & fuse the bacteria to produce multiple viruses
- The process by which viruses transfer genetic info from one bacterium to another is called TRANSDUCTION
4
Q
What viruses are used to express recombinant DNA?
A
- Non-primate lentiviruses are used to insert DNA into mammalian cells
- Baculoviruses are used to insert DNA into a eukaryotic system such as insects
5
Q
Where are the artificial chromosomes for recombinant technology taken from?
A
- The artificial chromosomes are taken from yeast. Known as YAC - Yeast Artificial Chromosome
6
Q
**What are 6 features of plasmid vectors?
A
- Plasmid vectors are relatively small (4-5Kb)
- Plasmid vectors can be linearized at multiple sites in non-essential stretches of DNA
- Plasmid vectors can have DNA inserted into them
- Plasmid vectors can be modified to have high copy number
- Plasmid vectors can be recircularised without losing the ability to replicate
- Plasmids contain selectable markers (antibiotic resistance markers)
7
Q
**How are recombinant proteins produced from recombinant DNA?
A
- Recombinant DNA transformed into E.Coli
- Placed on an agar plate containing antibiotic
- Recombinant plasmids should produce colonies
- Colonies can be isolated & cultured
- Protein can then be purified
8
Q
**Why are plasmids used as recombinant tools?
A
- Plasmids can increase the functionality of a gene
1. Expression of a recombinant gene
2. Add or modify control elements
3. Alter the properties of genes
9
Q
Give examples of 5 recombinant proteins for clinical use?
A
- Factor XIII - Hemophilia
- Insulin - Type 1 Diabetes
- Interferon - Viral Hepatitis
- Erythropoietin - Kidney disease & Anaemia
- Tissue Plasminogen Factor - Stroke & Embolism
10
Q
What are the requirements for cloning a gene in a bacteria?
A
- Must be able to replicate in bacteria
- Must be easy to replicate
- Must have a high copy number
- Must have a modified origin of replication
- Must be easy to manipulate
- Multiple cloning sites
11
Q
What 3 control elements are required for expression in bacteria?
A
- SHINE-DALGARNO SEQUENCE - a sequence of 8 bases upstream from AUG
- TRANSCRIPTION TERMINATOR
- BACTERIAL PROMOTER
12
Q
What are the two types of promoters?
A
- Constitutive
2. Inducible
13
Q
What is a constitutive promoter?
A
- A CONSTITUTIVE PROMOTER is always on, so it can express proteins to a high level
- However, this can be damaging if the protein is toxic
- It is also energetically expensive
14
Q
*What is a inducible promoter?
A
- An INDUCIBLE PROMOTER acts as a molecular switch
- They allow large cultures to be grown, without expressing the recombinant protein
- Even if the protein is toxic, it will have very little effect on the culture growth
- Inducible promoters use transcriptional repressors which can bind & dissociate
15
Q
What are the requirements of a DNA insert that is to be placed in a prokaryotic system?
A
- DNA must be easy to manipulate
- Must be a copy of the coding sequence
- Must include a start & stop codon
- Must not have introns as bacteria can’t splice
- Shouldn’t have a cap site, polyadenylation signal or eukaryotic UTRss
