Gene Cloning

Question 1

How has the concept of a gene changed over the past 150 years

Answer 1

• Up to 19th century: traits are inherited as “characteristics”
• Offspring receive a “characteristic” from each parent. E.g. pink flower from mating of a red one and a white one (blending inheritance)
• 1866 Mendel: rules to explain inheritance of biological characteristics
• Characters are defined by ‘elements’: discrete particles that don’t blend (Mendelian inheritance)
• These particles will later be called genes

Question 2

Explain how DNA came to be understood as the main key repository of genetic information

Answer 2

• 1928 Griffith- discovered bacterial ‘transformation’
• Started from observation of different bacterial strains
• 1944 avery, Macleod and McCarty – the transforming compound is DNA
• 1952 Hershey and Chase- DNA not proteins are the ‘genetic material’
• Used labelled bacteriophages to identify that DNA had entered cells

Question 3

Original cloning

Answer 3

• Original cloning involve taking a twig from a plant, planting it and it growing

Question 4

Molecular cloning

Answer 4

• Cloning a gene means isolating an exact copy of a fragment of DNA from an organism
• Involves copying the DNA sequence of that gene into a smaller, more accessible piece of DNA e.g. plasmid
• Start from chromosome, piece of dna introduced to vector
• Vector can reproduce itself and contain identical copies of the dna
• Traditional gene cloning:
• DNA is purified from a cell
• Fragment of the dna that contains a gene of interest is isolated using a restriction enzyme or PCR
• The dna fragment is inserted into a circular dna molecule, vector, in this case a plasmid to produce a recombinant DNA molecule
• Transform host cells with the vector
• When host cell divides copies of recombinant dna are passed to progeny and there is further vector replication
• After large number of cell divisions a colony or clone of identical host cell is produced
• Identify clone

Question 5

Sub-cloning

Answer 5

• Taking a clone from bacterial culture
• Transfer dna from one plasmid to another

Question 6

Main reasons to clone genes

Answer 6

• To obtain pure sample of an individual gene separated from all other genes in the cell
• Determine nucleotide sequence of specific genes
• So specific dna can be amplified
• So protein function can be investigated
• Medicine
• Agriculture
• Research
• Forensic science

Question 7

• Define the meaning of recombinant plasmid in the context of DNA cloning

Answer 7

• A fragment of DNA is inserted into plasmid DNA using DNA ligase

Question 8

• Outline the main steps used to clone DNA from any organism into bacterial vectors and create a dna library

Answer 8

• Foreign dna is digested with a restriction enzyme
• Bacterial plasmids are cut with same restriction enzyme
• A fragment of dna can thus be inserted into the plasmid dna using dna ligase to form a recombinant dna molecule
• *not all dna is genes
• Incorporate plasmids int bacterial honest cells by transformation
• Each cell contains different segment of dna from the original organism – DNA library
• A dna library is a collection of vectors containing lots of different types of insert
• Use libraries for screening – look for a piece of dna that you want
• Cells can now be plated out on agar medium
• Colonies of cells (clones) containing the desired gene can then be identified and isolated

Question 9

• Describe how restriction enzymes are used to splice DNA into cloning vectors

Answer 9

• First you have to isolate dna from an organism/cell that contains gene of interest
• Lyse cells by chemical, enzymatic and physical methods (e.g. sonication, homogenisation)
• Have to be careful or shearing dna as shearing forces used to break up dna
• Can use liquid nitrogen on plants and fungi to fracture cells open
• Remove membrane lipids with detergent
• Remove proteins by adding a protease/ phenol/ denaturing
• Remove rna with rnase (in practical we used NaOH as RNA is sensitive to base catalysed hydrolysis)
• Precipitate dna with alcohol
• Then
• DNA is fragmented with restriction enzymes (endonucleases) e.g. EcoRI, HindIII, etc. and DNA is cut into small pieces

Question 10

List the essential components of a plasmid cloning vector

Answer 10

• Could also use bacteriophage vectors or bacteriophage derived vectors but we only really use plasmids now
• Plasmid dna consists of:
• Origin of replication – not a gene (but still useful!)
• Antibiotic resistance gene
• Multiple cloning site (MCS) – contain lots of different restriction sites, artificial and inserted into plasmid. Designed as a series of restriction sites close together. Unique in that plasmid (restriction site doesn’t occur anywhere else in plasmid)
• Cleavage at any of these sites linearises the plasmid

Question 11

Use of plasmid cloning vectors

Answer 11

• Real cloning vectors are complex and highly engineered
• E.g. pBluescript SK
• SK due to orientation of MCS
• Allows people to make transcripts of dna as it contains bacteriophage promoter regions that can be used with bacteriophages to make transcripts of dna downstream from promoter
• Also contains sequences that can be used to prime sequencing reactions, e.g. PCR
• Usually carry a gene for drug resistance and a gene to distinguish plasmids with and without inserts

Question 12

• Explain how DNA ligases are used to clone DNA and the fundamental DNA “ligation” reaction

Answer 12

• Different dna pieces cut with the same restriction enzyme can join or recombine
• Restriction enzymes create staggered cuts in specific sequences to produce sticky or blunt ends
• Sticky ends hybridise
• DNA ligation to stabilise the dna as thr new strand now has H bonds and a covalently bonded backbone
• Ligase needs atp
• ATP binds active site then active site binds dna
• 5’ end must be phosphorylated
• Nucleophilic attack by 3’ end
• Ligase catalysed formation of phosphodiester bond

Question 13

Single RE to minimise vector re-ligation

Answer 13

• Single restriction enzymes:
• Non-directional – fragments generated can go in either orientation
• 50% chance of each orientation
• Self-ligation of the vector is a problem as it is more efficient than ligation of the insert
• The vector needs to be dephosphorylated to minimise self-ligation
• Use alkaline phosphatase (shrimp alkaline phosphatase (SAP) or calf-intestinal alkaline phosphatase (CIP)
• Because shrimps grow at very low temperatures so can easily inactivate SAP by raising temp. Stops insert being phosphorylated

Question 14

• Directional cloning – two different restriction enzymes to minimise vector re-ligation

Answer 14

• Would cut at non-complementary restriction sites
• Do same to vector and insert
• That’s why its good to have an MCS as many restriction sites
• Orientation is determined
• Self-ligation of the vector is prevented

Question 15

Blunt end ligation to minimise vector re-ligation

Answer 15

• Used when compatible restriction sites are not available
• Universal compatibility
• Self-ligation of vector
• Slower than sticky-end ligation

Question 16

Use of pcr in classical cloning

Answer 16

• Can introduce sequences in the primers in the 5’ end with enough complementarity to 3’ end and PCR will still work
• Also use primers with a restriction site in their 5’ end so they can be cut and ligated
• Don’t have to rely on existing sequences
• PCR requires some sequence information about 2 regions of dna of interest to synthesise the appropriate primers
• Primers are oligonucleotides complementary to different regions on the 2 strands of dna template (flanking the region to be amplified)
• Primers 15-20 nucleotides
• One hybridising to one strand of dsDNA, the other hybridising to the other strand such that both primers are oriented with their 3’ ends pointing towards each other
• Primer acts as a starting point for dna synthesis
• The oligo is extended from its 3’ end by dna polymerase

Question 17

Exploiting terminal transferase activity of taq polymerase

Answer 17

• Exploring ‘terminal transferase’ activity of Taq polymerase (adds an A on to the end) i.e. non-template polymerase acitivity
• Can be exploited by creating vectors that have a T overhang and getting them to ligate (T/A cloning)
• Ligase not very efficient
• Topoisomerase bound to vector
• Does ligation effectively into active site
• Uses vaccinia virus topoisomerase instead of T4 dna ligase (t/a topo cloning)
• Other variants exist (blunt/ restriction enzyme generated ends)

Question 18

Electroporation

Answer 18

• DNA can be introduced into the bacterial cells through the pores created by an electric field
• High effiency of transformation
• Put bacteria and plasmids between electrodes

Question 19

Cacl2/ heat shock

Answer 19

• The cells become competent when incubated with CaCl2 in cold condition, due to changes of the cell surface structure thus making it more permeable to dna
• The heat-pulse creates a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through pores
• Go through series of warm cold cycles

Question 20

Transformation into non-bacterial cells:

Answer 20

• E.g. introducing new dna into animal, fungal and plant cells
• For plants and fungi- the cell wall is removed (protoplasts)
• Precipitation of dna onto cell surface with Ca phosphate
• Introduction by liposomes
• Micro injection i.e. inject dna into nucleus
• Electroporation
• Biolistics (transformation with micro projectiles (can be done with cell walls intact)
• Introducing to bacterial:
• Plasmid dna can remain in circular shape in bacteria
• Plasmid dna replicates in rolling circle way
• Thus linear plasmid dna cannot be replicated

Question 21

Exploiting antibiotic resistance

Answer 21

• E.g. ampicillin resistance
• Ampicillin is an irreversible inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls
• Ampicillin causes cell lysis by inhibiting bacterial cell wall synthesis
• Beta-lactamase provides antibiotic resistance by breaking the beta lactam antibiotics structure such as ampicillin
• Beta lactamase gene can often be called ampicillin resistance gene or simply Amp^R
• E.g. kanamycin resistance
• Kanamycin interacts with the 30S subunit of prokaryotic ribsomes
• Kanamycin induces mistranslation and indirectly inhibits translocation during protein synthesis, thus causing cell death
• Aminoglycoside 3’-phosphotransferase: inactivates by phosphorylation a range of aminoglycoside antibiotics such as kanamycin
• Aminoglycoside 3’-phosphotransferase gene can often be called kanamycin resistance gene, or simply kan^R
• After transformation cells are plated onto ampicillin medium containing antibiotics (e.g. ampicillin/amp)
• All colonies are transformants (have a plasmid with amp^R)
• Untransformed cells give no colonies

Question 22

LacZ and blu/white:

Answer 22

• A plasmid vector that contains the lacZ gene which codes for part of the enzyme beta-galactosidase (alpha fragment)
• Some strains of e.coli have a modified lacZ gene (omega fragment)
• Bacteria will only synthesise the enzyme when a plasmid which harbours the missing lacZ segment is present
• Substrate is colourless, product reacts with itself to give an analogue of indigo, a blue compound
• White colonies are clones with a plasmid with the insert
• Bacteria with vectors with no insert are blue
• Insert disrupts the lacZ gene so colonies with the insert will be white

Question 23

Use of suicide genes

Answer 23

• If you put an insert in sacB you disrupt the gene for levansucrase
• Can select cells that are able to grow on sucrose

Question 24

Explain the challenges posed by “classical” restriction enzyme & ligation mediated cloning

Answer 24

• May be no appropriate restriction sites
• Use of ligase may be problematic – doesn’t always work well
• PCR has issues if what you are trying to clone has internal restriction site

Question 25

List four alternative developments in molecular cloning

Answer 25

• Ligation/ sequence and ligation independent cloning (LIC/SLIC)
• Gibson assembly
• Gateway cloning
• Golden gate

Question 26

SLIC

Answer 26

• Inserts amplified by PCR
• Vectors linearised by restriction enzymes or PCR
• 3’-5’ exonuclease activity of T4 DNA pol is exploited to create complementary sequences designed in primers
• Acts as exonuclease in absence of dNTPs
• Top strand is degraded starting from 3’ end
• When a dNTP is added the enzyme will stall, e.g. if dCTP is added it will stall at C
• Complementary sequence is treated with T4 pol
• Ends will hybridise
• Strong as it is a long piece of dna hybridising
• Long overlaps hybridise- then introduced to e.coli by transformation
• E.coli repair mechanisms join the ‘nicked’ pairs

Question 27

Gibson Assembly

Answer 27

• Design and create fragments with identical sequences at ends to be joined (e.g. by PCR)
• Create 5’ overhangs by digestion with T5 exonuclease (digests 5’ to 3’), chews back the dna
• Repair missing bases by DNA pol (e.g. Phusion- heat resistant polymerase), fills in gaps between annealed fragments
• Ligate with Taq ligase (also heat stable)
• All done at 50 degrees
• T5 survives at 50 degrees for a while before it is degraded
• Phusion pol then takes over and fills in any gaps
• All happens in one tube at one temperature
• Very simple
• Can assemble lots of fragments at the same time
• Multiple inserts, in directed order, can be assembled
• All fragments can be put in same tube at same time
• Vectors can be simply prepared by restriction enzyme digestion and repair
• Can assemble Oligos and make your own sequences

Question 28

Gateway cloning

Answer 28

• Relies on recombination rather than hybridisation of restriction cut ends and ligation
• Exploits the sequence specific recombination systems of e.coli and lambda bacteriophage
• Used for libraries
• Expensive because of the need to purchase LR- or BP- recombinase

Question 29

Golden gate assembly

Answer 29

• Alternative to gateway cloning
• Uses Type IIS restriction enzymes (different to type II in other cloning methods)
• Type IIS cut outside the recognition sequence
• You can design sequences and add them by PCR
• You can design religation however you want
• Not determined by recognition sequence
• Prepare fragment with recognition sequences either side so you lose them when you make the cut
• No recognition site in insert so can digest and ligate at same time
• Can have large numbers of ligations occurring at same time

Question 30

Pros and cons golden gate assembly

Answer 30

• Pros:
• Easy to use
• High flexibility of annealing sites as independent of recognition sites
• Cost effective
• Restriction-ligation cycles are irreversible
• Moderate number of type IIS REs are now available commercially
• Cons:
• Need to check that type IIS sites are not present in inserts and vectors

Question 31

Contrast the different approaches and identify how elements of “classical cloning” are incorporated into each of the above

Answer 31

• Gibson and SLIC both use exonuclease activity
• Gateway and golden gate are very similar and both use destination vectors
• Gibson is ligated similarly to classical
• Golden gate uses restriction enzymes, just a different type to classical
• In gibson assembly similar to classical in that vectors are prepared with restriction enzymes

Question 32

Discuss the caveats of PCR-based approaches to cloning and how these are dealt with

Answer 32

• Any cloning/sub cloning protocol that includes rounds of DNA polymerisation has a non-negotiable risk of introducing mutations
• Depending on downstream applications it may be essential to confirm that clones are not mutated
• Long-range sequencing e.g. nanopore can be used effectively to sequence entire plasmids

Question 33

How is rna cloned outline

Answer 33

• Extract RNA
• 1st strand cDNA synthesis
• 2nd strand cDNA synthesis
• Prepare ‘ends’
• Clone into appropriate vector
• Transform/package + infect
• Library

Question 34

First strand synthesis of rna cloning

Answer 34

• Eukaryotic mRNA has polyA tail
• Add oligo polyT primer
• Add dNTPs and reverse transcriptase which works 5’ to 3’
• Makes copy and gives double stranded heteroduplex of RNA and cDNA
• Can also add random primers
• Really short stretches of dna (6-8bp)
• Bind to RNA and enable RT
• End up with lots of small bits of cDNA that you can ligate
• Useful if no polyA tail
• Also good at priming targets that are very distant from polA tail as RT can fall off or become inhibited by secondary structure

Question 35

Second strand synthesis in rna cloning

Answer 35

• RNase cleaves RNA
• Add e.coli dna pol I that recognises cleaved parts and can polymerise dna
• Remnants of mRNA serve as primers for synthesis of the second strand of cDNA
• Complementary strands with nicks can be ligated

Question 36

CDNA directional cloning

Answer 36

• I.e. adding different adaptors to the 5’ and 3’ ends
• Prime 1st strand synthesis with an oligo containing RE site
• Second strand synthesis including some methylated dCTP
• Ligate ‘adaptors’ with specific RE site different to earlier RE
• Cut with 1st RE-any internal site will not be cut as protected by methylation
• CDNA will be directional i.e. RE site for second RE is at 5’ end of original RNA

Question 37

Critically evaluate the costs of cloning native DNA/cDNA and gene synthesis approaches

Answer 37

• DNA synthesis 10p per base
• CDNA synthesis kit is £3000 and you have to pay people to work for you to do the synthesis

Question 38

• List examples of fields in which Gene Cloning is applied

Answer 38

• Research tool
• Agriculture
• Medicine
• Forensic science

Question 39

Explain how Gene Cloning may be used in fundamental research

Answer 39

• E.g. production of transgenic organisms to study biological questions e.g. transgenic mice
• Transgenic mouse contains additional artificially introduced genetic material in every cell
• Used to study gene function/ regulation – gain of function e.g. mouse may produce a new protein or loss of function if the integrated dna interrupts another gene
• Transgenic mouse is a useful system for studying mammalian genes because analysis is carried out on the whole organism
• Transgenic mice also used to model human diseases that involve the over – or mis- expression of a particular protein
• Useful in studying gene function/regulation and to model human diseases caused by dominantly acting mutant proteins e.g. Alzheimer’s disease

Question 40

• Summarise how Gene Cloning transgenic mice may be used to investigate biological processes in mammals

Answer 40

• Can model human disease
• E.g.
• Normal mice cannot be infected with polio virus since they lack the cell-surface molecule (CD155) that in humans is the receptor for the virus. So normal mice can’t serve as a model for studying the disease
• Transgenic mice expressing the human gene for the polio virus receptor can be infected by polio virus —> paralysis and other pathologies similar to human disease

Question 41

Transgenic organisms

Answer 41

• Transgenic mouse has ‘random’ insertion into genome
• Can’t control where it is put
• May affect gene expression or cause mutation
• Mutation may not be due to sequence that you put but because of the sequence you disrupted

Question 42

Knock-out organisms

Answer 42

• Can either introduce a mutant, inactive version of a gene or delete the gene of interest all together
• Can do homologous recombination or CRISPR where foreign dna is inserted into desired locus replacing a portion of the original genome
• Or can grow ES cells allowing the growth of cells containing the DNA construct in a selective media as construct will have drug resistance marker
• ES cells containing the gene are introduced to early mouse embryo
• Some offspring have the mutation in germ line cells
• Can mate with normal mouse so offspring all have one copy of normal gene
• Can mate these offspring together to produce a mouse with both copies of target gene mutated
• Technique can be used to determine the function of genes

Question 43

Transgenesis using ES cells

Answer 43

• Embryonic stem (ES) cells are derived from very early mouse embryos and can differentiate into all types of cell when introduced into another embryo
• DNA—> ES cells may integrate randomly but use of CRISPR technology ensures a much higher chance of targeted insertion
• CRISPR – cas9 uses a guide rna molecule to direct it to the target sequence where it then introduces a double-strand break in the dna
• Guide rna allows it to search the genome and bind to the target of interest
• Target gene can be cut out and replaced by modified gene of interest
• ES cells will colonise host embryo and contribute to germ line —> production of some sperm varying the extra DNA
• When these transgenic sperm fertilise normal egg —> transgenic mouse with same foreign DNA in every cell

Question 44

Knock-in organisms

Answer 44

• Gene of interest is not deleted
• Additional function (e.g. mutation, chimera with foreign protein) is added

Question 45

Genetically modified maize to increase resistance to corn borer

Answer 45

• Example 1: BT corn
• European corn borer eggs are laid on the underside of leaves
• Hatch in 3-9 days depending on weather conditions
• Evades the effects of insecticide
• Solution: express insecticide directly in the plant
• Late-stage larvasse commonly tunnel into the ear shank of corn
• Cloning and expression of delta-endotoxin
• Bacterium bacillus thuringiensis(BT) has evolved defence mechanism to survive in the gut of insects by producing delta-endotoxin, CrylA(b)
• Accumulates as an inactive precursor in bacteria but after ingestion by insect the protoxin is cleaved by proteases in alkaline condition —> active toxin which binds to the epithelium of insect gut and causes cell lysis by the formation of cation-selective channels, which leads to death
• The protoxin cannot be cleaved in human gut, due to the high acidity in stomach
• CrylA(b) protein is 1115aa but toxic activity resides in segment 29-607
• Ligated into a vector between promoter and polyadenylation signal (required for production of mature mRNA for translation) from cauliflower mosaic virus
• Introduced into maize embryos by microprojectile bombardment

Question 46

Golden rice

Answer 46

• Vitamin A deficiency is a serious problem in developing world, responsible for 1-2 million deaths, 500,000 cases of irreversible blindness annually
• Golden rice was genetically engineered to express beta-carotene, a precursor of vitamin A, in the edible parts of rice (rice plants can naturally produce beta-carotene in leaves, where it is involved in photosynthesis)
• Project started In 1993
• In July 2021 the Philippines approved it for commercial propagation

Question 47

Identification of gene causing cystic fibrosis

Answer 47

• Autosomal recessive genetic disorder
• Causes difficulty in breathing, sinus infections, poor growth, infertility
• Characterised by abnormal transport of chloride and sodium ions across an epithelium, leading to thick, viscous secretions
• Cystic fibrosis is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel that transports chloride ions
• CFTR regulates the movement of chloride ions across epithelial membranes.
• Mutations in CFTR gene lead to loss of function, resulting in accumulation of chloride ions inside the cells, causing sticky mucus to build up on the outside of the cells
• Cystic fibrosis is a good canditate for gene therapy as it is caused by mutations in a single gene
• A modified common cold virus was used as a vector to carry the normal CFTR gene to the cells in the airways of the lung
• One study of liposome-based CFTR gene transfer therapy demonstrated some improvements in respiratory function in people with CF but this limited evidence of efficacy does not support this treatment as a routine therapy at present
• No evidence of efficacy for viral-mediated gene delivery

Question 48

• Forensic identification:

Answer 48

• Any type of organism can be identified by examination of DNA sequences unique to that species
• Identifying individuals within a species is less precise but as DNA sequencing technologies develop direct comparison of very large DNA segments (whole genomes) are possible —> precise individual identification
• DNA profiling: molecular genetic analysis that identifies DNA patterns
• Restriction fragment length polymorphism (RFLP) can be studied against Variable number tandem repeat (VNTR)
• Perform southern blotting using a probe for the repeat