DNA cloning and modification Flashcards
Give 4 major applications of genetic modification.
- Protein production, e.g. insulin
- Research into gene function by creating knock-out mutants
- Food production, GM crops and animals
- Gene therapy
When was the first recombinant DNA created and by who?
1972, Paul Berg’s group
What was the first recombinant DNA a hybrid of?
The SV40 and Lambda viruses
Define recombinant DNA.
DNA that has been formed artificially by combining constituents from different organisms.
What is the principle of clonal growth and how is it used in recombinant DNA?
Bacteria grow by dividing into exact clones. You insert the recombinant DNA into a plasmid and as the bacteria grow they produce huge volumes of exact copies.
Are plasmids essential to bacterial survival?
No.
What kind of genes do plasmids contain?
Additional, non-chromosomal DNA.
Plasmids usually provide specific functions that allow bacteria to be well-adapted to their environments. Give 3 examples of plasmid function.
- Nitrogen fixation
- Antibiotic resistance
- Virulence (‘the degree of pathogenicity of a microbe’)
Plasmids can be transferred between bacteria, true or false?
True, by conjugation.
Transmission rate of plasmids between bacteria is high. True or false?
False - rate of transmission is very low at ~1%.
What can increase the rate of transmission of plasmids between bacteria? Give 3 examples.
- Heat shock
- Electroporation
- Application of particular chemicals
Give an example of a chemical that can make a bacterial cell more receptive to transformation.
Calcium chloride, CaCl2.
What was the first transgenic organism and when was it created?
Xenopus DNA was inserted into bacterial plasmids in 1973.
Who created the first transgenic organism in 1973?
Cohen and Boyer.
What is the basic principle of creating recombinant DNA?
Digest 2 pieces of DNA with the same restriction endonuclease so they have complementary sticky ends, then join them together in ligation.
Plasmids contain an origin of replication. What does this mean?
A particular sequence in the plasmid where replication is initiated.
Define a multiple cloning site (MCS).
A section of DNA that contains multiple restriction endonuclease recognition sites.
What is a polylinker?
Another name for a multiple cloning site.
Multiple cloning sites are typical of engineered plasmids. Why?
Restriction sites typically only occur once within a plasmid.
Multiple cloning sites are often flanked by promoters/regulators etc. Why?
To regulate endonuclease activity.
What do multiple cloning sites allow?
DNA insertion.
How many restriction sites per RE are there in each MCS?
1, but there may be others outside the MCS.
Marker genes are often inserted into plasmids, for example resistance or fluorescent protein genes. Why?
Allows you to identify which plasmids have been transformed - those that do not display the marker gene have not uptaken the plasmid.
What does the LacZ gene encode?
β-galactosidase.
What is β-galactosidase?
A hydrolase enzyme that catalyses the hydrolysis of β-galactosidase into monosaccharides.
β-galactosidase mutants are missing something. What is it?
An α-peptide at the N-terminus.
Define α-complementation.
Whereby the α-peptide is restored in LacZ mutants.
The functional β-galactosidase gene has an effect on X-Gal. What is it?
It turns colourless X-Gal blue.
What is X-Gal?
A colourless lactose analogue.
Why is α-complementation used in transformation experiments?
To see which bacteria have uptaken the gene - the mutants that turn the substrate blue have been transformed back to wild type.
Define a bacteriophage.
Viruses that infect bacteria.
Explain briefly how bacteriophages work, give 6 steps.
- Phage attaches to bacteria and injects chromosome
- Bacterial chromosome is degenerated by phage enymes
- Phage chromosome is replicated using bacterial machinery
- Phage genes expressed to produce new phage components
- Progeny phages assemble
- Bacterial cell wall split by lysis, progeny phages released
What is M13?
A filamentous bacteriophage.
How many closely-packed genes does it have for infection and replication?
10.
M13 vectors can accommodate large inserts of DNA. True or false?
False - they only have room for very small inserts, up to 3kbp.
M13 phage vectors are often engineered to include lacZ markers and MCS. True or false?
True.
Lambda phages can accommodate larger inserts than M13 phages. True or false?
True.
What is the size of inserts limited by in Lambda phages?
The amount of DNA that can be packaged into a capsid, between 38-51kbp.
Define a capsid.
The protein coat of a virus.
What is a cosmid?
A hybrid of a plasmid inserted with Lambda phage cos sequences.
What do cos sites allow?
Packaging of DNA into phage capsids.
Cosmids allow larger inserts than normal plasmids. How big can they be?
Approx. 45kb as opposed to 15kb of normal plasmids.
What is an artificial chromosome?
Synthetic chromosomes made of fragments of DNA inserted into a host chromosome.
What is the point of an artificial chromosome?
They are introduced into host cells to propagate and can be used to transfect other cells.
Give 2 examples of artificial chromosomes.
- BAC = bacterial artificial chromosome
2. YAC = yeast artificial chromosome
What is the maximum insert size for a BAC?
Approx. 350kbp
What is the maximum insert size for a YAC?
Up to 2000kbp.
Put in order 6 possible vectors, with number 1 that accommodates the smallest inserts and number 6 with the largest.
- M13 phage
- Normal plasmid
- Lambda phage
- Cosmids
- BAC
- YAC
What is a vector in terms of DNA?
A way of transmitting DNA from one organism to another.
Give 2 advantages of using bacteria to amplify target DNA.
- Cheap and self-propagating
2. Do not need to know the sequence as you do with PCR
What is splicing and why does it happen?
The removal of introns (non-coding DNA) from mRNA transcripts. Splicing ensures only the exons of genes are transcribed.
Splicing occurs in both eukaryotes and prokaryotes. True or false?
False - it only occurs in eukaryotes as prokaryotes do not contain introns.
You cannot properly express eukaryotic DNA in bacteria. Why?
Because it contains introns.
How is it possible to overcome the problem that eukaryotic DNA is not properly expressed in bacteria?
Use eukaryotic mRNA to make cDNA using reverse transcriptase, as mRNA has been spliced so the introns have been removed.
Where do reverse transcriptases come from?
Retroviruses.
Define a DNA library.
A mixture of recombinant DNA clones that represent a significant proportion of the complete genome. Basically so we have a record of all the genes in an organism.
DNA libraries can be of 2 types. What are they?
- Genomic DNA
2. cDNA (complementary DNA)
What is the point of a DNA library?
Used in genetic screening, DNA preparation and gene expression analysis.
When generating a DNA library, larger inserts are better. Why?
Because then fewer clones are required, as each one will contain a larger percentage of the whole genome.
Complete and partial digests are used in the generation of DNA libraries. Why?
To create overlapping fragments that can be re-aligned during analysis.
How do you search a DNA library for a particular gene? Give 2 methods.
- Make an ssDNA labelled probe that will attach to the sequence of interest
- Use antibodies labelled with dye
Define a microarray.
A set of DNA sequences representing the entire set of genes (not entire genOME) in an organism, arranged in a grid pattern for genetic testing.
