Recombinant DNA Technology Flashcards
What is a gene library?
Large collections of cloned DNA fragments, representing all/most of the genes present in the organism.
What are the 2 types of gene clone?
DNA and mRNA.
What is a limitation of DNA based gene cloning?
It cannot be used in bacterial cells - contains introns.
What is important about the mRNA used to generate cDNA?
It must be from a tissue that expresses the gene being cloned.
What is the function of oligo-dT primer and reverse transcriptase in producing cDNA?
To generate a single strand of cDNA from the mRNA.
What is the function of RNAse H and DNA polymerase in producing cDNA?
To generate double stranded cDNA which is blunt ended.
Why is genetic cloning important commercially?
Difficult to obtain proteins can be made in large quantities by expression in GM organisms.
What is reverse genetics?
Determining the phenotype that results from the mutation of cloned genes.
What can be determined from cloning?
The nucleotide sequence, the amino acid sequence and the evolutionary history of the organism.
Why are cloned genes used as probes?
They can determine in which tissues and at what developmental stages, the cloned gene is expressed.
How do restriction enzymes work?
They recognise and cleave at specific points of DNA sequences.
What are the 2 types of ends produced from restriction enzyme action?
Sticky ends and blunt ends.
What word describes restriction enzyme cutting sites?
Palindromic - the top and bottom strands have the same sequence.
How long are the DNA sequences that restriction enzymes target?
Usually 4bp or 6bp.
What is the function of DNA ligase?
It makes covalent bonds between two different DNA fragments.
Does DNA ligase work on blunt and sticky ends?
It can join both but blunt ended fragments are joined with reduced efficiency.
What are the key features of a plasmid vector?
One or more unique restriction enzyme sites.
An origin of replication.
A selectable marker.
What is a selectable marker in terms of a plasmid vector?
A feature that allows cells with the plasmid to be distinguished from cells that lack it.
Why is E.coli commonly used in commercial protein production?
E.coli is easily manipulated and can be easily grown on a large scale.
Which codons are identical in both prokaryotes and eukaryotes?
Initiation (AUG) and termination (UGA) codons.
What are the 3 problems associated with expressing eukaryotic genes in E.coli?
- Eukaryotic genes contain introns.
- Eukaryotic promoters/terminators become non-functional.
- Eukaryotic mRNAs lack ribosome binding sites.
How can you overcome E.coli being unable to remove introns?
Obtain the eukaryotic gene from a cDNA library - cDNA lacks introns.
How can you overcome eukaryotic promoters becoming non-functional in prokaryotes?
Insert the cDNA from the eukaryotic gene into an expression vector which contains a prokaryotic promoter/terminator either side of the gene.
How can you overcome eukaryotic mRNA lacking ribosome binding sites?
Use an expression vector that also contains a ribosome binding site before inserting the cDNA.
What is the Shine-Dalgarno sequence?
A ribosome binding site in prokaryotes.
How do you perform gel electrophoresis?
- Take a slab of agarose and pipette DNA/RNA into wells.
- Apply an electric field, DNA/RNA migrate towards positive electrode.
- Smaller fragments move faster through the gel.
- Stain to view bands.
What is Sanger sequencing used for?
To sequence a single gene or DNA fragment.
What is next generation sequencing used for?
To sequence a whole genome or millions of fragments.
What is required for Sanger sequencing?
Dideoxynucleoside triphosphates (ddNTP) and DNA polymerase.
Why are dideoxynucleoside triphosphates (ddNTP) used in Sanger sequencing?
When incorporated into a growing DNA chain, they terminate DNA synthesis. This is due to no 3’-OH being present.
How can specific mRNA/DNA fragments be identified in a mixture?
Using a probe that can base pair (hybridise) only with the complementary fragment.
What molecules and probes are involved in northern blotting?
Molecules on gel = RNA
Probe = labelled DNA or RNA
What molecules and probes are involved in southern blotting?
Molecules on gel = DNA
Probe = labelled DNA or RNA
What molecules and probes are involved in western blotting?
Molecules on gel = proteins
Probe = antibody
What are the 3 basic steps of ‘blotting’?
- Separate molecules via gel electrophoresis.
- Blot molecules onto membrane.
- Incubate membrane with probe and expose.
What is northern blotting used for?
To determine when and where a gene is expressed.
What is PCR?
Targeted amplification of a specific gene sequence.
What are the uses of PCR?
Gene cloning, measuring gene expression, DNA profiling and disease diagnosis.
What are the requirements for PCR?
2 oligonucleotide primers and a heat stable DNA polymerase.
What is are oligonucleotides?
Short fragments (17-25bp) of single stranded DNA.
What is the forward primer in PCR?
Same sequence as a stretch of of the 5’ end of the top DNA strand.
What is the reverse primer in PCR?
Corresponds to the 5’ end of the bottom DNA strand (complementary to the 3’ end of the top DNA strand).
What does a PCR reaction contain?
DNA being amplified, primers (in excess), mixture of nucleoside triphosphates and DNA polymerase.
What are the 3 steps in PCR and what temperatures are they performed at?
Denaturation of template DNA = 95ºC
Primer annealing = 45-65ºC
Incubation (DNA synthesis occurs) = 72ºC
How long is each PCR cycle?
30-60 seconds.
How does PCR produce so much DNA?
It is an exponential cycle, every cycle it doubles.
Which part of the sequence undergoes exponential amplification?
Only the sequence between (and including) the binding sites for the primers.
What is the mechanism of DNA fingerprinting based on?
STRs - short tandem repeats.
Why are STRs used to identify individuals?
The number of STRs varies greatly between individuals and occur at many sites.
How is a DNA profile generated?
PCR is carried out using primers that recognise non-varying DNA either side of an STR.
What is a transgenic animal?
An animal that has exogenous DNA inserted into its genome.
What are the benefits of AquAdvantage salmon?
Increased growth rate and diet utilisation.
What is used to produce transgenic plants?
The Ti (tumour inducing) plasmid.
How is the Ti plasmid used to make transgenic plants?
It can be used as a vector to introduce exogenous DNA into plants.
What bacterium is the Ti plasmid found in?
Agrobacterium.
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats.
What actually is CRISPR?
An adaptive immune system found in bacteria and archaea.
What happens when a bacteriophage infects a bacterium?
Small fragments of bacteriophage DNA are inserted into bacterial genome at the CRISPR locus.
Transcription of fragments produces crRNA forming a complex with tracrRNA and Cas9.
What is Cas9?
A DNAse enzyme.
What happens if the bacterium is reinfected by the same bacteriophage?
Cas9/tracrRNA/crRNA complex recognises the bacteriophage DNA and cleaves it.
What are the two mechanisms by which double stranded breaks are repaired?
Homology directed repair and non-homologous end joining.
How accurate is homology directed repair?
Very accurate.
How accurate is non-homologous end joining?
Error prone.
When can homology directed repair operate?
Only in S-phase and early G2-phase.
When can non-homologous end joining operate?
In any phase of the cell cycle.
