Genetic manipulation Flashcards
What is a clone?
A DNA molecule/ cell/ organism that is genetically identical to the DNA molecule/ cell/ organism from which it is derived
Give an overview of gene cloning.
- Obtain DNA from organism of interest
- Break into fragments or amplify gene of interest by PCR
- Ligate into vector
- Introduce into bacterial cells
- Identify bacteria with a plasmid carrying the gene of interest. Grow cells and purify plasmid DNA
What are some applications for gene cloning in research?
Determine nucleotide sequence to analyse evolutionary history of gene and species
Use clone as probe to find when gene is expressed, helps learn about diseases
Test the biological function of the gene with reverse genetics
What is reverse genetics?
Manipulate the organism so the gene is either over-expressed or mutated so non-functional to find the purpose of the gene
What are some applications of genetic manipulation in medicine?
Can determine underlying cause of disease
Can develop diagnostic tests and sometimes new therapies
What are the applications of genetic manipulation in biotechnology?
If the gene encodes a pharmaceutically or commercially important protein it can be inserted into a host and expressed to produce lots of the protein
DNA profiling
Altering crops to have characteristics beneficial to us like disease resistance
What do restriction enzymes do?
Recognise and cleave at specific DNA sequences, commonly 4 or 6 base pairs in length
What is a key feature of restriction enzyme cutting sites?
Palindromic: the top and bottom strands have the same sequence
What enzyme can make covalent bonds between two different DNA fragments that have the same sticky end? What else can this enzyme do, just less efficiently?
DNA ligase
Can also join blunt ended fragments together, but this ligation is less efficient
Define ligation.
Joining of two DNA strands of other molecules by a phosphate dieter linkage
Where can DNA be inserted using restriction enzymes and DNA ligase?
Into a plasmid or other vector
What is a vector for DNA cloning? What are the most commonly used vectors?
A DNA molecule that carries the exogenous DNA fragment into a host cell in which the vector can replicate
Modified plasmids are the most commonly used vectors
What are some host cells used in cloning?
E. coli
Other types of bacterium
Yeast
What does exogenous mean?
Growing or originating outside an organism
Name 3 key features of plasmids used as vectors in gene cloning.
- One or more unique restriction enzyme sites in which DNA can be ligated
- An origin of replication
- A selectable marker that allows cells with the plasmid to be distinguished from cells that lack it (usually an antibiotic resistance gene)
What are some other desirable features of a plasmid vector? (2 features)
- High copy number
2. A means of distinguishing recombinant plasmids (e.g. with exogenous DNA inserted) from non-recombinant plasmids
Restriction enzymes cleave DNA at …
These sites are normally … in length and are …
Specific recognition sites
4bp or 6bp in length
Palindromic
What are two types of gene clone?
Genomic clones
cDNA clones
What is a genomic clone?
Cloning of chromosomal DNA generates genomic clones
Must be used if the promoter or intron-exon structure is to be analysed as this is not in cDNA
Cannot be used to make proteins in E. coli as it can’t splice out introns
What is a cDNA clone?
Cloning cDNA (DNA complementary to mRNA) generates cDNA clones cDNA clones must be used if the protein made by the gene is to be produced by recombinant bacteria
Why are E. coli often used to produce valuable proteins with genetic manipulation?
Easily manipulated and can be grown on a large scale
What are some issues that must be overcome to express Eukaryotic genes in E. coli cells?
Introns: bacterial cells can’t remove introns. So obtain the genes from a cDNA library as this comes from reverse transcription of mRNA so lacks introns
Eukaryotes and prokaryotes don’t use the same promoters and terminators. Use expression vectors
Prokaryote will need a ribosome binding site and eukaryotic mRNAs don’t have this. Use an expression vector that also contains a ribosome binding site
What is an expression vector?
Introduces a specific gene into a target cell that contains a prokaryotic promoter and terminator either side of the inserted cDNA
The expression vector used will also often have a ribosome binding site immediately before the site of cDNA insertion
The protein insulin is made in which host organism?
E. coli
What genetically manipulated host organism is used to produce human growth hormone?
E. coli
What host organism makes chymosin? What is chymosin?
E. coli, Kluyveromyces lactis, fungi
Protein used in cheese making
What host organism is used to make hepatitis-B vaccine?
Yeast
Name the host organism used to make herceptin. What does herceptin do?
Cultured mammalian cells
Used to treat some cancers by blocking HER2 and encouraging the immune system to attack and kill cancer cells
What host organism is Antithrombin III produced in?
Transgenic goats
What is gel electrophoresis?
A method to separate DNA or RNA molecules according to their size.
What are the gels in gel electrophoresis?
Slabs of agarose and polyacrylamide
Describe gel electrophoresis.
DNA or RNA is pipetted into wells made at one end of gel
Apply electric field. DNA and RNA migrate to positive electrode
Gel is porous so acts as sieve. Smaller fragments migrate faster
Gel is treated with stain allowing individual DNA/ RNA fragments to be seen as distinct 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 thousands or millions of DNA fragments
How are DNA fragments identified in sequencing?
Identified as they pass through a fluorescence detector
Automated process
How has sequencing helped fight disease?
Has led to the identification of many mutations that cause disease
What does our Lord and Saviour (Glen Sweeney) refer to as finding a needle in a haystack?
Detecting specific nucleic acid sequences
How can specific mRNA or DNA molecules in a mixture be identified?
By using a probe that can base-pair (hybridise) with then
The probe hybridises only to the complementary fragment
What are blotting methods used for? What are three types?
Blotting methods are used to detect specific molecules present in a mixture that has fractionated by electrophoresis
Southern blotting, Northern blotting, Western blotting
What is each type of blotting used to detect?
Southern blotting: DNA
Northern blotting: RNA
Western blotting: Proteins
What is the purpose of Northern blotting?
To determine when and where a gene is expressed
What is in situ hybridisation and when is it used?
Whole embryo or segment of tissue incubated with digoxygenin (labelled probe made from gene of interest)
The probe hybridises to mRNA from the gene of interest that is present in the embryo or in the section on the slide
Detect bound probe with antibody that recognises digoxygenin
What does PCR stand for?
Polymerase chain reaction
What is PCR?
The targeted amplification of a specific DNA sequence
Using this the amount of a chosen DNA fragment can be increased a billion-fold or more in 2-3 hours
When can PCR be used?
Cloning of genes
Measurement of gene expression
DNA profiling
Diagnosis of genetic and pathogen-induced disease
What are the two key requirements for PCR?
- Primers. These are oligonucleotides: short fragments of single stranded DNA that are chemically synthesised. Two primers are needed: one to base pair with each end of the DNA fragment being amplified. The primers make PCR specific: they determine which DNA fragment is amplified
- A heat stable DNA polymerase. This is usually Taq DNA polymerase from the bacterium Thermus aquaticus
What are the two primers needed for PCR?
Forward primer: same sequence as a stretch of the 5’ end of the top strand of the DNA to be amplified
Reverse primer: corresponds to 5’ end of bottom strand of the DNA fragment to be amplified (complementary to 3’ end of top strand)
What does a PCR reaction contain?
- A small amount of the DNA being amplified.
- The primers
- A mixture of dATP, dCTP, dGTP and dTTP.
- Taq DNA polymerase
How many cycles does PCR usually involve?
20-40 cycles of amplification
What are the three steps of each cycle of PCR?
- Incubation at 95 degrees celsius to denature the template DNA
- Incubation at 45-65 degrees celsius to allow primers to anneal to the single stranded DNA
- Incubation at 72 degrees celsius. Optimum temp for Taq DNA polymerase so DNA synthesis occurs during this step
How long does each step in PCR take?
30-60 seconds
What is exponentially amplified in PCR?
Only the sequence between and including the binding sites for the primers undergoes exponential amplification
Describe what DNA profiling is.
DNA profiling allows individuals to be unambiguously identified. It relies on differences between the genomes of different individuals. It is based on differences in short repeated sequences known as short tandem repeats (STRs).
What is a short tandem repeat?
Short repeated sequence that DNA profiling is based on
They occur at many sites in the genome of humans and other animals
The number of copies of the repeat in an STR varies greatly between individuals
How to obtain a DNA profile.
PCR is carried out using primers that recognise sites in the non-varying DNA either side of an STR
Size of the PCR products depends on the number of repeats
Each persons DNA produces two PCR products from their two alleles, with the number of repeats varying between alleles and person to person
This makes a different pattern of PCR products
How do we increase the degree of discrimination in PCR?
Several STRs would usually be tested from each person involved
Why is PCR accurate enough for use in forensic science?
It is so sensitive only a small spot of blood, or some dandruff, etc is needed to provide enough DNA to generate a DNA profile
DNA profiles can link a suspect to a crime
A large database of DNA profiles of known offenders has been assembled
In criminal investigations, more than 10 STRs are used as other than twins, no two people should match the same for this
How is DNA profiling used for paternity testing?
For each STR, a child inherits 1 allele (band on gel) from the mother and 1 from the father
What diseases can PCR be used to diagnose?
Huntington’s disease
Most other genetic disorders like Cystic Fibrosis and sickle cell disease
Also infections, HIV
How is PCR used to diagnose Huntington’s disease?
PCR is performed using primers that flank the trinucleotide expansion
The size of the PCR product depends on the number of CAG repeats that are present
Isolate DNA from patients => PCR => electrophoresis
This test can be used in pre-natal diagnosis
It can be used to test adults with an affected parent to tell them whether they will go on to develop Huntington’s disease
What is a transgenic animal?
An animal that has exogenous DNA inserted into its genome
What are transgenic animals used for?
Produce pharmaceutically/ commercially important proteins by generating transgenic animals that secrete the protein into their milk
“Improve” farm animals
Research
Using pronuclear microinjection, what percentage of offspring will be transgenic?
25%
How many copies of the DNA are injected to a single site?
1-200 copies
What are some commercial applications of additive transgenesis?
In aquaculture, AquAdvantage salmon. Makes them grow at a faster rate and improves utilisation of their diet
Poduction of valuable proteins in transgenic animals
Transgenic plants with resistance to pests and herbicides and more nutritional properties
What are some concerns of AquAdvantage salmon?
Human health- altered levels of hormones/ allergens?
Environment: escaped transgenic fish may breed with wild population
What is the Ti plasmid?
Tumour inducing plasmid
How does Agrobacterium cause plant tumours?
It contains the Ti plasmid
The Ti plasmid contains a region of DNA that promotes tumour formation
The T region becomes integrated into the genome of the infected plant cells
How can the Ti plasmid be used to make transgenic plants?
The Ti plasmid can be used as a vector to introduce exogenous DNA into plants susceptible to infection by Agrobacterium
What global proportion of cotton plants are genetically manipulated?
80%
What global proportion of soybean plants are genetically manipulated?
77%
What global proportion of maize plants are genetically manipulated?
32%
What was the estimated economic gain for 1996-2016 due to crop plants being genetically manipulated?
$186.1 billion
What is CRISPR?
Tool for editing genomes adapted from the natural defence mechanisms of bacteria and archaea
What are double stranded breaks in eukaryotic cells repaired by?
Homology directed repair
Non-homologous end joining
What does homology directed repair do?
Accurately repairs double-stranded breaks, but only operates in S-phase and early G2-phase of cell phase
What does non-homologous end joining do?
Repairs double-stranded breaks, in any phase of the cell-cycle but is error prone
What can happen following cleavage by CRISPR?
Following cleavage by CRISPR, DNA repair may lead to inactivation of the target gene, or can be exploited to make a precise change in the target gene.
