Gene Cloning - Lecture Flashcards

Question

What is the antibiotics resistance gene motif in plasmid DNA?

Answer 1

Antibiotics resistance genes * Code proteins exhibit resistance to antibiotics * Act as selectable markers to identify bacteria with a particular plasmid i.e. in the presence of ampicillin, only cells expressing the protein for antibiotic resistance can grow • Most popular antibiotic selections: Ampicillin & Kanamycin

Answer 2

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. β-lactamase (produced by bacteria) provides antibiotic resistance by breaking the β-lactam ring in the antibiotics' structure such as ampicillin. β-lactamase gene can often be called ampicillin resistance gene, or simply ampR

Answer 3

Kanamycin interacts with the 30S subunit of prokaryotic ribosomes. Kanamycin induces substantial amounts of mistranslation and indirectly inhibits translocation (movement between compartments) during protein synthesis, thus causing cell death --\> basically messing up protein synthesis. Aminoglycoside 3'-phosphotransferase (also known as Neomycin phosphotransferase II) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as kanamycin --\> inhibits kanamycin by phosphorylation. Aminoglycoside 3'-phosphotransferase gene can often be called kanamycin resistance gene, or simply kanR

Answer 4

After transformation cells are plated onto ampicillin medium → colonies All colonies are transformants (harbours a plasmid with ampR --\> hence they shall survive). Untransformed cells are ampicillin-sensitive → no colonies

Answer 5

A plasmid vector that contains lacZ gene which codes for part of the enzyme beta-galactosidase. Some strains of E. coli have a modified lacZ gene & only synthesise the enzyme when a plasmid which harbours the missing lacZ segment is present. There are multiple cloning site is in the middle of LacZ gene. Important ---\> Insertion of an external DNA fragment into MCS causes disruption of LacZ gene, thus no functional b-galactosidase expressed, resulting white colony – blue/white selection --\> which allows for identification.

Answer 6

1. To achieve gene over-expression or knock out to study the function of that gene 2. For the production of recombinant proteins in mammalian cells 3. Gene therapy to treat human disease

Answer 7

Bacteriophages (λ phage) - Genes encoding head & tail proteins & other genes involved in host cell lysis are clustered in distinct regions in ~ 50 kb l phage genome - Genes irrelevant for survival/growth can be deleted from the phage genome & replaced by other DNA sequences of interest. - Insert size is limited to ~ 25 kb due to the requirement that the DNA has to fit into the phage head

Answer 8

Viruses as cloning vectors For example..... - Adenoviruses: will take DNA fragments up to 8kb. Induce inflammatory genes e.g. IFNg in a transduced cell. - Papillomaviruses: High capacity for inserted DNA. Does not cause death of infected mouse cells & BPV molecules are passed to daughter cells → permanently transformed cell line - Adeno-associated viruses\*: Inserts into host DNA at same position within human chromosome 19 - Retroviruses (e.g. Lentivirus)\*: commonly used in gene therapy. Inserts at random positions → very stable integrants • \*potentially useful in gene therapy

Answer 9

- The wild-type lentiviral genome is made up of packaging genes (gag, pol), envelope gene (env), and long-terminal repeats (shown in black) - The genome can be separated into 3 plasmids, packaging, envelope, and integrating vector for transgene (converting the viral genome into 3 plasmids) - The 3 plasmids are transfected into a cell line to produce the viral vectors, which can be collected from the supernatant.

Answer 10

YAC (yeast artificial chromosome) YACs are hybrids of bacterial plasmid DNA & yeast DNA --\> Components required for replication/segregation of natural yeast chromosomes combined with E. coli plasmid DNA.

Answer 11

- YACs are grown in yeast (Saccharomomyces cerevesiae) & contain selectable markers --\> selectable markers allow the growth of transformant on selective media lacking specific nutrients (non-transformants unable to grow). Note --\> Yeast strains used are auxotrophic i.e. unable to make a specific compound --\> Trp1 mutants make no tryptophan only grow on media supplemented with tryptophan But if transformation takes place --\> YAC will contain intact Trp1 gene --\> able to grow on media. The problem --\> YACs are unstable and frequently lose parts of the DNA during propagation

Answer 12

YAC libraries useful for cloning very large DNA fragments (\> 1 Mb) cloning large genes (e.g. 250kb cystic fibrosis gene) & creating libraries of large overlapping clones e.g. for individual chromosomes (chromosomal libraries)

Answer 13

A genomic library is a collection of clones sufficient to contain every single gene present in a particular organism.

Answer 14

A collection of cloned cDNA (complementary DNA) fragments from particular cells or tissues. cDNA is produced from mRNA, thus contains only the expressed genes of cells/tissues --\> Hence.... It does not contain untranslated region, e.g. promoters, introns.

Answer 15

Genomic library is prepared by isolating total DNA from the organism, digesting it into fragments of suitable size & cloning them into a vector i.e. shotgun cloning. Subsequently, one has to introduce the vectors into a suitable host. Then one can screen, identify and characterise the different clones Lastly, we plate the clones onto a plate and maintain them there --\> known as a genomic library. Basically.... Genomic libraries are prepared by: 1. Extracting & purifying total cell DNA 2. Making a partial restriction digest 3. Cloning DNA fragments into a suitable vector 4. Transform bacteria with recombinant DNA 5. Characterize the library

Answer 16

To clone fragments of genomic DNA which are even as small as 20kb we cannot just cut the DNA to completion with a restriction enzyme --\> thus partial digestion is used. Because.... - 4 bases in recognition site of enzyme → average distance between sites --\> 256 - 6 bases in recognition site of enzyme → average distance between site --\> 4096 Neither of these distances is long enough to be useful in complete digestion so partial digestion is used to generate suitable large fragments of DNA --\> May get non-random distribution of fragment sizes due to non-random distribution of enzyme sites.

Answer 17

Isolation & ligation of DNA fragments 1. Purify fragments of required size by agarose gel electrophoresis & elute fragments from gel 2. Ligate fragments into a vector pre-cut with a restriction enzyme using DNA ligase

Answer 18

**Prokaryotes** (smaller genomes) – can make gene libraries in plasmids --\> Plasmid inserts are roughly 5-10 kb so only need a few thousand recombinants for a representative library. **Eukaryotes** (larger genomes) need vectors that can hold much larger insert DNA fragments i.e λ phage.

Answer 19

Screening by colony (or plaque) hybridisation 1. Phage particles & extracellular viral genomic DNA transferred to a nitrocellulose filter. 2. Nitrocellulose filter is incubated with a radiolabelled (or biotinylated) probe for the gene of interest. 3. The probe will hybridize with the gene of interest. 4. Autoradiograph will locate the clone with the gene of interest --\> identify colony(s) that express gene of interest. 5. Clone can be isolated & inoculated into new host cells for further amplification

Answer 20

- Probe - cloned piece of DNA containing portion of sequence for which you are searching. - The probe will bind to any clone containing sequences similar to those found on the probe --\> This binding step – hybridization - Probe can be cDNA, gene from another organism, related gene, oligonucleotide, PCR fragment etc

Answer 21

- Multi-cellular organisms have specialization of individual cells e.g. liver cells, brain cells etc --\> Each cell contains the same set of genes but in different cell types different sets of genes are switched on while others are silent - Hence, if messenger RNA (mRNA) cloned only those genes being expressed will be cloned --\> If mRNA is the starting material then resulting clones comprise only a selection of the total number of genes in the cell - mRNA can be cloned as cDNA

Answer 22

No, mRNA cannot be cloned directly but a DNA copy of the mRNA can be cloned - This conversion is accomplished by the action of reverse transcriptase & DNA polymerase 1. Reverse transcriptase makes a single-stranded DNA copy of the mRNA 2. Second DNA strand is generated by DNA polymerase & double- stranded product is introduced into an appropriate plasmid or l vector

Answer 23

- Key to cDNA cloning procedure is synthesis of cDNA from mRNA template by reverse transcriptase 1. mRNA obtained & purified from other RNAs by trizol extraction & column purification Note - Reverse transcriptase cannot initiate DNA synthesis without a primer 2. Use oligo(dT) as primer – this is complementary to poly(A) tail at the 3'-end of most eukaryotic mRNAs --\> Oligo(dT) binds to poly(A) at 3'-end of mRNA & primes DNA synthesis using the mRNA as a template. 3. reverse transcriptase can synthesize cDNA Note - Random primers can also be used

Answer 24

1. After mRNA copied → single-stranded DNA ("first strand") the RNA is partially degraded with ribonuclease (RNase) H1. Degrades RNA strand of RNA/DNA hybrid. 2. Remaining RNA fragments act as primers for DNA pol1 "second strand synthesis" which uses the first DNA strand as the template --\> Result is double-stranded cDNA

Answer 25

1. Ligate cDNA into a vector --\> cDNAs have no sticky ends so ligate blunt ends or attach sticky ends (for more efficient ligation) 2. cDNA clones are representative of mRNA present in the original preparation 3. cDNA library would contain a large proportion of clones representing insulin mRNA (other clones will also be present) 4. Identify clones by hybridization of specific probe

Answer 26

1. **Research tool** --\> e.g. production of transgenic animals to study biological questions 2. **Agriculture** --\> e.g. genetically modified crops such as increased resistance of maize to corn borer 3. **Medicine** --\> e.g. bulk production of compounds such as factor VIII, gene therapy etc 4. **Forensic science** --\> e.g. identification of crime suspects --\> DNA profiling

Answer 27

Research tools: 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 the analysis is carried out on the whole organism --\> Transgenic mice also used to model human diseases that involve the over- or misexpression of a particular protein Example of use... - Normal mice lack the cell surface receptor (CD155) --\> can't be infected by the poliovirus --\> however, transgenic mice expressing the CD155 gene can be infected --\>

Answer 28

1. Foreign DNA introduced → gain of function e.g. production of a new protein or the expression of an existing protein at a higher level or in a different range of cells. 2. Useful in studying gene function/regulation & to model human diseases caused by dominantly acting mutant proteins e.g. Alzheimer's disease 3. Removing normal function of gene --\> loss of function --\> interrupts/disturbs the expression of an existing gene.

Answer 29

1. Inject DNA of interest into pronucleus (Pronucleus - sperm cell becomes a pronucleus after the sperm enters the ovum but before the genetic material of the sperm and egg fuse (also applies for egg)) 2. Gene should be flanked by a promoter and Poly A --\> promoter is needed because the place of insertion is random and random promoter that gene gets may be weak. 3. Inject pronucleus into oocyte 4. Allow oocyte to develop 5. Transgenic mice created.

Answer 30

The process used --\> Homologous recombination --\> Inserts foreign DNA into desired locus replacing a portion of the original genome 1. Obtain + culture Embryonic stem cells --\> can differentiate into any cell 2. Gene we constructed present in a vector --\> is homologous to an actual gene (picture --\> red gene flanked by yellow regions) --\> endogenous gene is targetted for elimination (yellow gene in the picture) --\> inserted into ES cells 3. Enzyme recombinase is then used to swap genes ---\> original DNA becomes destroyed This is can occur randomly but if introduced DNA is similar in sequence to part of mouse genome it may undergo 'homologous recombination' & integrate as a single copy at a specific site 4. Include antibiotic resistant gene into the inserted gene --\> so we can isolate specific cells by examining antibiotic resistance 5. Targetted cells can then be injected into early embryo --\> Embryo implanted into the uterus --\> note these ES cells will colonise host embryo & contribute to the germline --\> results in the production of some sperm carrying the extra DNA 6. Mate chimeric mouse with normal to give transgenic offspring --\> because when these transgenic sperm fertilise normal egg --\> produces transgenic mouse with foreign DNA in every cell

Answer 31

Similar process Replace gene with another functional gene which has a single mutation --\> analyze gene by point by point mutation Adding fluorescents to analyse the function with homologous recombination.

Answer 32

Transgenic: DNA is injected into zygote --\> i.e. without homologous recombination. Knockout/in: DNA is injected into ES cells --\> i.e. with homologous recombination

Answer 33

Transgenesis: the process of introducing an exogenous gene (transgene) into an organism, so that the organism will transmit that exogenous property to its offspring.

Answer 34

1. Genetically engineered crops --\> e.g. replacement for insecticides 2. Cloning of genes in animals --\> e.g. transgenic farm animals for protein production 3. Cloning of animals --\> e.g. Dolly

Answer 35

BT corn - Cloning & expression of δ-endotoxin Pesticide --\> European corn borer --\> ruin corn --\> eggs are laid on the underside of leaves --\> evades the effects of insecticides. - Bacterium Bacillus thuringiensis (BT) has evolved defence mechanism to survive in the gut of insects by producing δ-endotoxin, CryIA --\> δ-endotoxin is a protoxin meaning that once it is ingested it is cleaved by proteases in alkaline condition --\> it can then bind to the epithelium of insect gut and causes cell lysis by the formation of cation-selective channels, which leads to death . Note --\> the protoxin cannot be cleaved in human gut, due to the high acidity in stomach. - CryIA(b) protein is 1115 amino acids in length but toxic activity resides in segment 29-607 therefore first 648 codons made by PCR. - Ligated into a vector between promoter & poly-A signal from cauliflower mosaic virus - Introduced into maize embryos by microprojectile bombardment

Answer 36

Context --\> 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 β-carotene, a precursor of vitamin A, in the edible parts of rice. - Golden rice was created by transforming rice with these two genes which result in the production of two enzymes (phytoene synthase and crt1) which are needed to convert geranylgeranyl-PP into Lycopene which can then be converted to alpha-carotene and beta-carotene by an enzyme which is already present (Lycopene cyclase)

Answer 37

- Production of recombinant pharmaceuticals e. g. insulin, growth hormone, vaccines etc - Identification of genes causing human diseases e. g. cystic fibrosis etc - Gene therapy & cancer e. g. cystic fibrosis, congenital blindness RPE65 defect etc

Answer 38

Factor VIII is a protein that plays a central role in blood clotting --\> Failure to synthesize factor VIII leads to haemophilia. - Until recently it was treated by injection of purified factor VIII --\> but purification difficult to remove virus particles that may be present. - However, using gene cloning methods we can produce factor VIII in animals (hamsters) which can then be given to people that suffer from the condition. - Factor VIII 186 kb --\> 2351 amino acid polypeptide --\> protein requires post-translational processing to form dimeric protein with 17 disulphide bonds. Hence...... It is cloned as 2 subunits –\> each cDNA fragment ligated into an expression vector between promoter and polyadenylation signal.

Answer 39

**Cystic fibrosis** Cystic fibrosis is an autosomal recessive genetic disorder --\> Causes difficulty in breathing, sinus infections, poor growth, and 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 --\> basically chloride channel doesn't pump out ions (ions stay inside cell instead) --\> causing sticky mucus to build up on the outside of the cells

Answer 40

- To give an indication of the biochemical basis to the disease enabling therapies to be designed - Identification of a mutation can be used to design a screening programme to identify carriers or those who have yet to develop the disease - Development of gene therapy

Answer 41

The basic concept of gene therapy involves introducing gene into a target cells to cure, prevent or slow down the progression of disease. Examples: 1. Manipulation of cells removed from an organism which are transfected & placed back e.g. stem cells from bone transfected with DNA used in the treatment of blood disorders 2. Cancer cell gene therapy → anti-sense RNA to silence oncogene

Answer 42

Congenital blindness - One type of congenital blindness is caused by a mutation in the Retinal Pigment Epithelium-specific 65 kDa protein (RPE65) - RPE65 processes a type of vitamin A needed to keep light-sensing photo-receptor cells (i.e. the rods and cones of the retina) in operating order --\> when mutated it no longer performs this functions resulting in blindness. - Administration of an adeno-associated virus (AAV) carrying the normal RPE65 gene was injected into the eyes of the patients, aiming to replace the non-functioning RPE65 gene with one that works --\> this restored vision for the majority of patients. -

Answer 43

- Any type of organism can be identified by examination of DNA sequences unique to that species. - DNA profiling: molecular genetic analysis that identifies DNA patterns --\> Based on the principle that individuals have their unique 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

Gene Cloning - Lecture Flashcards

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