recombinant DNA technology

Question 1

vocab - defintions for
*Recombinant DNA
*Genetic manipulation / genetic engineering
*Clone
*Gene cloning

Answer 1

*Recombinant DNA - DNA that has been formed artificially by combining two or more fragments from different sources.
*Genetic manipulation / genetic engineering - directed or pre-determined alteration of genotype of an organism.
*Clone - DNA molecule/cell/organism that is genetically identical to the DNA molecule/cell/organism from which it was derived.
*Gene cloning - is a biological method of purification. DNA from the gene of interest can be inserted into a “vector” (often a plasmid) which is then introduced into a host cell. When the vector replicates, the inserted DNA is also replicated, allowing large amounts of it to be isolated.

Question 2

Outline the importance, and main experimental and commercial applications of gene manipulation technologies.

Answer 2

Analyse the cloned gene (sequencing, determination of expression profile etc.).
Where relevant, determine if gene is involved in inherited diseases-develop diagnostic tests, therapies
Produce recombinant protein and/or pure mRNA by in vitro transcription
Make specific mutations in the gene in vitro
Introduce into plants (generate transgenic plants)
Introduce into animals (generate transgenic animals)
Make precise changes in genes in vivo, e.g. using CRISPR

Question 3

applications of genetic manipulation

Answer 3

A research tool:
Once a gene has been cloned, Its nucleotide sequence can be determined.
From this:
1. The amino acid sequence of the encoded protein can be deduced.
2. The evolutionary history of the gene and its host species can be analysed.
3. Primers for PCR can be designed.

The clone can be used as a probe, e.g. to determine in what tissues, and at what developmental stages, or disease states, the cloned gene is expressed.

Devise experiments to understand the function of genetic material or genes. This can include manipulating the organism from which it is derived so that the gene is either over-expressed, or mutated so as to be non-functional (reverse genetics).

Medicine:
Once a gene has been cloned:
Genes involved in genetic diseases, or in cancer, 
disease-associated mutations within the gene can be 
identified.
This allows:
*The molecular basis of the disease to be determined.
*The development of diagnostic tests, and sometimes new therapies (Some RNA or DNA based), for the disease.
*Techniques to rapidly sequence entire genomes allow:
(i) The genetic changes in cancers cells to be identified.
(ii) Foetal DNA present in the plasma of a pregnant woman to be analysed. (Basis of non-invasive pre-natal testing for chromosomal abnormalities).

biotechnology:
Pharmaceutically or commercially important protein can be expressed in a suitable host organism (e.g. E. coli or yeast) allowing large amounts of the protein to be manufactured
*DNA profiling in forensic science and paternity testing.
*Genetic manipulation of organisms (plants, animals, microbes)
*to give altered growth or disease resistance properties, improve nutritional value or enable bioremediation.
*to produce commercially important compounds or proteins

Question 4

Describe the reactions catalysed by restriction enzymes and the DNA sequences those enzymes recognise.

Answer 4

Restriction enzymes were discovered because the growth of bacteriophages is restricted in some types of bacteria. 
These bacteria make restriction enzymes that cleave 
DNA from infecting bacteriophages
*Restriction enzymes recognise, and cleave at, specific 
DNA sequences. These are commonly 4 or 6 base-pairs (bp) in length (Type II restriction enzymes)
DNA fragments produced by restriction enzyme digestion have either “sticky” or blunt ends
Restriction enzyme cutting sites are palindromic - the top and bottom strands have the same sequence (read 5’ to 3’)

Question 5

Describe the use of DNA ligase in gene cloning.

Question 6

Discuss the features of basic plasmid vectors used in gene cloning.

Answer 6

*DNA is cloned by inserting it into a vector – A DNA molecule that carries the exogenous DNA fragment into a host cell in which the vector (and the inserted DNA) can replicate.
*Modified plasmids are the most commonly used vectors

Host cells include:
E. coli (most routine cloning procedures)
Other types of bacterium
Yeast

Plasmids used as vectors in gene cloning must have:
*One or more unique restriction enzyme sites (i.e. only occurring once in the plasmid) into which DNA can be ligated. (Often designed to contain a multiple cloning site / polylinker region)
*An origin of replication specific to for the host organism.
*A selectable marker that allows cells with the plasmid to be distinguished from cells that lack it. Usually an antibiotic resistance gene.

Other desirable features of a plasmid vector:
*High copy number
*Small size (a few thousand bp)
*A means of distinguishing recombinant plasmids (i.e. with exogenous DNA inserted) from non-recombinant plasmids.

Isoschizomers - Enzymes which recognize same site and cuts in the same way
Neoschizomers - Enzymes which recognize same site but cuts differently (SmaI, XmaI)

Question 7

cloning a gene of interest

Answer 7

Clones of genes may be obtained from gene libraries –These are large collections of cloned DNA fragments, representing all or most of the genes present in the organism from which the library has been prepared.
Or
If you know the sequence the gene of interest can be amplified from genomic DNA or cDNA by PCR (or chemically synthesised).

Question 8

types of gene clone

Answer 8

Cloning of chromosomal DNA generates genomic clones.
*Genomic clones must be used if the promoter or the intron-exon structure is to be analysed since promoter and intron sequences are not present in cDNA .
*Genomic clones cannot be used to make protein in E. coli since it cannot splice out introns.

*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.

Question 9

productions of proteins in e.coli

Answer 9

*Protein that is otherwise difficult to obtain can be made in large quantities by expression in genetically manipulated organisms.
*A protein being manufactured by a genetically manipulated organism will usually represent >1% of all the protein made by the organism.
*E. coli are commonly used as to produce valuable proteins since they are easily manipulated and can be easily grown on a large scale.
*Other hosts for protein production include other bacteria, yeast, fungi, cultured mammalian cells, insect cells etc.

Question 10

Difficulties that must be overcome when expressing eukaryotic genes in prokaryotes

Answer 10

problem:
Bacterial cells cannot remove introns from 1° transcripts.
Solution:
Obtain the eukaryotic gene from a cDNA library. cDNA clones are prepared by reverse

problem:
The promoters and terminators of eukaryotic genes do not function in prokaryotic cells.
Solution:
Insert the cDNA from the eukaryotic gene into an expression vector that contain a prokaryotic promoter and terminator either side of the inserted cDNA.

problem:
In order to be translated a prokaryotic mRNA needs a ribosome binding site.
Eukaryotic mRNAs do not have ribosome binding sites.
Solution:
Use an expression vector that also contains a ribosome binding site immediately before the site of cDNA insertion.

Question 11

Explain the difference between cDNA and genomic DNA and describe preparation of double-stranded cDNA.

Question 12

 Discuss how bacteria may be manipulated in order to produce pharmaceutically or commercially important proteins.

Question 13

Poly Chain Reaction(PCR)

Answer 13

PCR is the targeted amplification of a specific DNA 
sequence. This may be a small fragment (e.g. a gene) present in a large population of DNA (e.g. a genome).
*The amount of a chosen DNA fragment can be 
increased a billion-fold or more in 2-3 hours.
*For example, starting with human DNA from a few cells, 
PCR could be used to amplify the b-globin gene until >10 copies of the gene are present

PCR may be used in:
Cloning of genes
Measurement of gene expression
DNA profiling
Diagnosis of genetic and pathogen-induced disease

Question 14

PCR requirements

Answer 14

To amplify a gene of interest you must know the sequence (at least of the ends or flanking regions)
*Target DNA
*Primers – 2 short segments of single stranded DNA – bind to sequence to enable synthesis to start
*A heat stable DNA polymerase - Taq DNA polymerase from the bacterium Thermus aquaticus
*dNTPs - a mixture of dATP, dCTP, dGTP and dTTP
*Buffer containing Mg2+ (cofactor for polymerase)
*Thermal cycler – computer programmed heating block – rapidly changes temperature

Question 15

PCR primers

Answer 15

*Oligonucleotides - usually 17-25 nucleotides.
*Two primers are needed – one to base-pair with each end of the DNA fragment being amplified.
*Primers make PCR specific - they determine which DNA fragment is amplified;
- must be of high specificity to reduce non-specific annealing
21nt oligonucleotide = 421 chance of occurring elsewhere
*Primers are present in huge excess compared to the fragment of DNA being amplified

Question 16

 Explain how a PCR reaction is assembled and discuss the importance of role of the primers and Taq DNA polymerase.

Question 17

Explain the events that occur during the denaturation, primer annealing and DNA synthesis steps of a PCR cycle.

Answer 17

PCR usually involves 20-40 cycles of amplification. Each cycle has three steps.

Denaturation - Incubation at 95°C (typically 30-60s) to denature DNA into single strands
Annealing - Incubation at 40-65°C (typically 30s) to allow primers to anneal to ssDNA
Extension - Incubation at 72°C (typically 1-3 min). Optimum temperature for Taq DNA polymerase so DNA synthesis can occur.

Experiment requiring 30 PCR cycles takes ~2-3 hours.

Question 18

PCR-polymerase

Answer 18

Polymerases have been continually developed and improved.
Versions of Taq DNA polymerase still widely used – cheap and efficient
*Problem = error prone ~285/106 bases mis-incorporated (E. coli DNA polymerase ~9/106)

Proofreading polymerases (e.g. Pfu Pyrococcus furiosus) - have RNAse H-like domain, therefore 3’- 5’ proofreading (also has longer half-life than Taq)
Reduces chance of errors, but more expensive
*Have to ensure dNTPs present prior to adding enzyme (Hot Start enzymes)
*(Occasionally we want error prone PCR)

Question 19

role of PCR primers

Answer 19

Primers can be designed to incorporate non-specific sequences.
*Sufficient specificity to target sequence and optimized PCR conditions enable amplified product to be made.
Enables incorporation of;
*new restriction sites
*tags for purification
*mutations in amplified sequence
*flanking sequences to enable homologous recombination (gene replacement)

Question 20

DNA synthesis for gene cloning

Answer 20

Recently become much more affordable, can have whole gene synthesised (~1.5 kbp) and cloned in a vector for ~£250 and delivered in less than 1 month.

Design DNA sequence exactly as you want it
*Exclude / include restriction sites
*Select codon usage (dependent on host to be used for expression)
*Can generate genes just from a sequence - no source DNA or cDNA required

Question 21

Explain how to make mRNA (e.g. for vaccines) in vitro.

Answer 21

Many plasmid vectors (e.g. pGEM3Z) include promoters for bacteriophage
RNA polymerases to allow transcription of inserted DNA in vitro.

eg. Making RNA vaccine against SARS-Cov2:
Insert gene encoding virus spike protein into suitable plasmid vector
Transcribe in vitro, with uridine replaced by pseudouridine (reduces non-specific immune activation and increases stability).
Add 5’ cap and 3’ poly A tail to RNA
Remove vector DNA, purify spike protein mRNA
Package pure spike protein mRNA into lipid nanoparticles
VACCINE

Question 22

Explain the basis of gel electrophoresis.

Answer 22

Agarose (polysaccharide, from seaweed) (0.5 - 2 % w/v agarose)
Seperates DNA and RNA molecules according to their size Enables
* Estimation of size of DNA fragments
* Monitoring of restriction enzyme cleavage
* Preparative isolation of cleaved DNA fragments
* Assessment of purity of DNA
* Investigation of conformations of DNA

• DNA (or RNA) is pipetted into wells made at one end of the gel.
• An electric field is applied - DNA and RNA migrate towards the positive electrode.
• Gel is porous - smaller fragments migrate faster than larger fragments.
  Rate of migration of linear molecules is inversely proportional to the log10 of their size.
• The gel is treated with a stain that allows individual DNA/RNA fragments to be seen as distinct bands on the gel.

Question 23

Outline methods to detect specific DNA and/or RNA molecules

Answer 23

Intercalating dye used to visualize DNA with UV
* Ethidium Bromide (EtBr)
* SYBR Safe
DNA ladder (marker) enables estimation of fragment sizes

Question 24

DNA profiling

Answer 24

• A Short Tandem Repeat (STR) is a sequence of 2-6p (e.g. TCAT) repeated from 1-50 times.
• DNA profiling relies on differences in STR repeats in individuals’ genomes.
• STRs occur at many sites in the genome of humans and other animals. e.g. an STR on chromosome 11.
• The number of copies of the repeat in a particular STR varies enormously between different individuals (and between the two alleles of the STR in the same individual).
• PCR using primers that recognise sites in the non-varying DNA either side of an STR. Different individuals generate a different pattern of PCR products.
• The size of the PCR products depends on the number of repeats.
  – Each person’s DNA produces two PCR products from their two alleles
  – The number of repeats varies between alleles and from person to person.

To increase the degree of discrimination, several STRs would usually be analysed from each person being tested.

Question 25

Explain how DNA profiles can be generated for forensic science
by using PCR to amplify tandem repeat sequences.
and paternity testing

Answer 25

PCR is very sensitive - a small spot of dried blood, or a flake of dandruff, provides sufficient DNA to generate a DNA profile
DNA profiles can be used to link a suspect to a crime – e.g. DNA from blood from a victim of a violent crime may be identified on clothing of a suspect.
In criminal investigations DNA profiles are assembled by analysing >10 STRs - no two individuals in the world (other thanidentical twins)shouldhavethesameDNAprofile.

paternity testing
For each STR, a child inherits one allele (band on gel) from the mother, one from the father.

Question 26

detecting specific amino acid sequences

Answer 26

Specific mRNA(orDNA)molecules in a mixture can be identified using a probe that can base-pair(hybridise) with them

northern blotting
Northern blotting is used to determine when and where a gene is expressed
Example - when during the development of a fish embryo does the gene encoding the muscle protein Troponin-T start to be transcribed?

Question 27

Describe quantitative real-time PCR (qRT-PCR) and its use in the diagnosis of COVID-19 infections.

Answer 27

Real-time PCR measures the amount of product formed in each cycle
qPCR - real time PCR / quantitative PCR (DNA template)
* Detect presence of DNA
* Quantify the number of copies of target DNA in a sample
qRT-PCR- real time/ quantitative reverse transcription PCR (cDNA template)
* Detect presence of cDNA – (only present if RNA template is present)
* Quantify the number of copies of target cDNA in sample
* Copies of virus (viral load) of infecting retrovirus eg. SARS-CoV2
* Measure mRNA levels (gene expression level) relative to control gene

• Amplification of DNA is monitored using a fluorescent dye or a sequence-specific DNA probe labeled with a fluorescent molecule.
• Fluorescence is measured after each cycle, allowing product accumulation to be monitored whilst the reaction is ongoing.
• Amplification of DNA is monitored using a fluorescent dye or a sequence-specific DNA probe labeled with a fluorescent molecule.
• Fluorescence is measured after each cycle, allowing product accumulation to be monitored whilst the reaction is ongoing.

sanger sequence applications:
* Sequence a single gene or fragment

Routine laboratory/research sequencing applications
* Check clones are sequence we expect
* Identify mutations in specific genes (PCR)

Question 28

measuring amplification

Answer 28

• The number of cycles required to reach a defined threshold quantity of product is determined (Cycle threshold = Ct).
• The more abundant the target sequence 0.5 at the start of the PCR, the fewer the cycles needed to reach the threshold.
• Can determine copy number of your target DNA or RNA in an unknown sample.
• Can compare to housekeeping gene(gene expression level)

Question 29

Outline some applications of next generation DNA sequencing.

Answer 29

Next generation sequencing applications
* Sequence whole genome or millions of individual fragments
Genome sequencing and resequencing: once
a reference sequence has been obtained for a species, genome sequences of different
individuals within that species can be
obtained rapidly.

Determining gene expression (transcriptomics).
Isolate mRNA from a tissue, convert to cDNA
and sequence. Data reveals which genes are
expressed and whether expression is high or
low.

Question 30

Explain how transgenic animals can be made.

Answer 30

transgenic animal - an animal that has exogenous DNA inserted into its genome. In biotechnology, transgenic animals may be made in order to:

• “Improve” farm animals.
  *Produce pharmaceutically/commercially important proteins by generating transgenic animals that secrete the protein into their milk.
  Transgenic techniques to alter a gene in the genome of certain animals are used for research purposes.

Making a transgenic animal by pronuclear microinjection

Question 31

Discuss the generation of AquaAdvantage salmon and transgenic goats that produce milk containing pharmaceutically valuable proteins.

Answer 31

Aquadvantage salmon:
Advantages:
*Increased growth rate (but not ultimately larger than normal).
* Improved utilisation of diet

Concerns:
*Human health - altered levels of hormones/allergens?
*Environment - escaped transgenic fish may breed with 
wild population.
Approved for sale in Canada and the USA

Question 32

transgenic plants

Answer 32

Transgenic plants (plants that have exogenous DNA insertedinto their genome) are generated with the aim of:
1. Manufacturing commercially important proteins.
2. “Improving” agricultural species, for example by:
*producing plants that are resistant to pes insects.
*producing plants that are resistant to herbicides.
*producing plants with improved nutritional properties.

Question 33

Explain how the Ti plasmid is used to make transgenic plants and give examples of the use of transgenic plants in agriculture.

Answer 33

*Infection of some plants by the bacterium Agrobacterium tumefaciens causes plant tumours (known as crown galls).

*Tumours arise because Agrobacterium contains the Ti (tumour inducing) plasmid.

*The Ti plasmid can be used as a vector to introduce exogenous DNA into plants susceptible to infection by Agrobacterium.

The Ti plasmid contains a region of DNA (the T region) that promotes tumour formation. During an infection by Agrobacterium the T region becomes integrated into the genome of the infected plant cells.

Question 34

commercial importance and concerns about transgenic plants

Answer 34

1. Bt Corn and cotton. Bacillus thuringiensis synthesises an insecticidal protein. The gene that encodes the toxin can be introduced into plants to make them resistant to pest insects. 

2. Herbicide resistant Soya. The herbicide glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate-resistant transgenic soya contains an EPSPS gene from a bacterium that makes EPSPS that is not affected by glyphosate.

Global proportion of crop plants that are genetically manipulated:
*Cotton: 80%
*Soybean: 77%
*Maize: 32%
*Estimated economic gain for 1996-2016: 
$186.1billion

concerns:
*Could herbicide resistant plants transfer their genes into
other species producing herbicide resistant “superweeds”?
*Might there be unforeseen effects of modification of the 
plant such that they become harmful to consumers?
Could plants encourage overuse of a single class of herbicide and emergence of resistance

Question 35

CRISPR

Answer 35

CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats
DNA sequences found in the genomes of prokaryotes - derived from DNA fragments of bacteriophages that had previously infected the prokaryote.

*Cas9 (CRISPR-associated protein 9) is a endocnuclease enzyme that uses CRISPR sequences as a guide to recognise and cleave specific strands of DNA that are complementary to the CRISPR sequence.

Isolated from streptococcus pyogenes

Re-engineered Cas9 to enable genome editing

An adaptive immune system from bacteria and archaea!

During a bacteriophage infection, small fragments of bacteriophage DNA become inserted into the bacterial genome at the CRISPR locus.

Transcription of these fragments produces CRISPR RNA (crRNA). This forms a complex with a trans-acting RNA (tracrRNA) and a DNAse enzyme (Cas9).

If the bacterium is subsequently infected by the same type of bacteriophage, the Cas9/ tracrRNA/crRNA complex recognises the bacteriophage DNA (due to base pairing with crRNA) and cleaves it.