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Genetic Engineering Flashcards

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1
Q

DNA cloning

A
  • copy of a specific gene
  • cloning a gene means isolating an exact copy of a single gene from the genome and copying it into a smaller more accessible factor (like a plasmid)
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2
Q

Purpose of DNA cloning

A
  • obtain a pure sample of a gene and determine its nucleotide sequence
  • by expressing this specific DNA/protein the protein’s function can be investigated
  • mutations can also be identified
  • ‘engineer’ organisms for our use
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3
Q

Overview of Cloning Process

A
  1. DNA purified from a cell. fragment of DNA containing a gene of interest is isolated using a restriction enzyme or PCR
  2. DNA fragment is inserted into a circular DNA molecule, vector, like a plasmid to make a recombinant DNA molecule
  3. transform host cells with the vector which then replicates producing numerous identical copies of itself and the genes it carries
  4. host cell divides to form copies of recombinant DNA passed to progeny: further vector replication
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4
Q

DNA Isolation

A

Isolate DNA from organism with the gene of interest:

  • lyse cell with physical or chemical methods (sonication or homogenisation)
  • remove membrane lipids with detergent
  • remove proteins with protease
  • remove RNA with RNase
  • precipitate DNA with alcohol
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5
Q

DNA digestion

A
  • use endonucleases to fragment DNA
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6
Q

Plasmid Structure

A
  1. origin of replication: DNA sequence ensuring the plasmid is replicated during the cell cycle
  2. antibiotic resistance gene
  3. multiple cloning site: series of restriction enzyme sites used to connect the fragment/gene to the plasmid
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7
Q

Single Restriction Enzyme

A
  • non directional (can’t tell which way the gene will be oriented)
  • self ligation of the vector can occur (plasmid reattaches to itself rather than the insert- this is more likely). the vector needs to be dephosphorylated to minimise this
  • alkaline phosphatases used to remove phosphate from the plasmid
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8
Q

Two Restriction Enzymes

A
  • directional cloning
  • no dephosphorylation needed
  • two endonucleases used to cleave plasmid leaving two complementary stick ends for the insert
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9
Q

Blunt end ligation

A
  • no hybridization of ends
  • non directional
  • need a phosphate treatment
  • is much slower than stick end ligation
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10
Q

DNA Fragment Extraction from Agarose Gels

A
  • cutting out target bands from agarose gels
  • melting agarose gels at around 50 degrees (lower than melting point of DNA)
  • purify DNA fragments using glass beads and silica: sodium ions attach to oxygen and attach to DNA forming a cation bridge. pure water treatment then releases the DNA
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11
Q

PCR Based Cloning

A
  • requires information about DNA region of interest to synthesise appropriate primers
  • primers are oligonucleotides complementary to different regions on the 2 strands of DNA template flanking amplification region
  • primers hybridize to one strand of the dsDNA and one to the other strand so both primers are oriented with their 3’ ends pointing towards each other
  • primers are the starting point for elongation at 3’ end by the DNAP
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12
Q

Insertion of Restriction Sites

A
  • uses PCR based cloning
  • need primer with homology to DNA
  • end of primer contains the restriction site
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13
Q

TA Cloning

A
  • uses PCR based cloning
  • can prepare a complementary plasmid for insertion without endonucleases
  • Taq DNA polymerases add adenine to the 3’ end of the product
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14
Q

Topoisomerase I Ligation

A

TOPO cloning:

  • Vaccinia virus topoisomerase I specifically recognises and digests DNA sequence (C/T)CCTT, and unwinds the DNA and re-ligates it again at the 3’ phosphate group of the last thymidine.
  • TOPO vectors carry (C/T)CCTT at the two linear ends. The linear vector DNA already has the topoisomerase enzyme covalently attached to both of its strands’ free 3’ ends.
  • PCR is performed to amplify target DNA fragment.
  • Once the PCR products and TOPO vectors are mixed, the topoisomerase catalyse ligation of the two ends at r.t. in 5 min.
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15
Q

Recombinase Ligation

A

Cloning by homologous recombination:

  • PCR is performed with primers containing overlap sequences with vectors.
  • DNA recombinases recognise overlapped sequences between vectors and PCR products, and catalyse the recombination and insertion of the PCR product into the vector.
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16
Q

Ligase

A
  • connects 3’OH to 5’P together

- uses ATP for energy

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17
Q

Electroporation

A
  • DNA introduced into cells through pores created by an electrical field
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18
Q

Heat Shock

A
  • cells become competent when incubated with CaCl2 in ice due to changes in the cell surface structure and increasing permeability to DNA
  • heat pulse creates thermal imbalance across membrane, allowing DNA entry through pores
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19
Q

Transformation in Non-bacterial cells

A
  1. precipitation of DNA onto cell surface with Ca phosphate
  2. introduction by liposomes
  3. transformation of plant protoplasts i.e. plant cell after wall has been degraded
  4. microinjection i.e. inject DNA into nucleus
  5. biolistics i.e. transformation with microprojectiles
  6. electroporation
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20
Q

Rolling Circle Replication

A
  • nick and displacement of nicked strand
  • replication by DNAP III creating ds plasmid
  • rejoining of nicked strand and synthesis of ds plasmid from ss circle
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21
Q

Antibiotic Resistance Genes

A
  • Code proteins exhibit resistance to antibiotics
  • Act as selectable markers to identify bacteria with a particular plasmid
    i. e. in presence of ampicillin only cells expressing the protein for antibiotic resistance can grow
  • Most popular antibiotic selections: Ampicillin & Kanamycin
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22
Q

Ampicillin

A
  • irreversible inhibitor of transpeptidases
  • cell lysis due to inhibition of cell wall synthesis
  • B-lactamase breaks B-lactam ring of antibiotics so this gene confers resistance
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23
Q

Kanamycin

A
  • interacts with 30S subunit of prokaryotic ribosomes
  • induces substantial mistranslation + indirectly inhibits translocation during protein synthesis (cell death)
  • neomycin phosphotransferase II phosphorylates and inactivates aminoglycoside antibiotics like kanamycin
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24
Q

LacZ screening

A
  • used to identify recombinants
  • uses plasmid with modified lacZ gene coding for only part of the B-galactosidase
  • enzyme only synthesised when plasmid containing missing lacZ segment is present
  • MCS is in the middle of the lacZ gene
  • DNA fragment insertion into the MCS disrupts lacZ gene so it is non functional
  • B-galactosidase hydrolyses X-gal into a blue precipitate
  • therefore, recombinants are recognised by their white color
25
Q

Types of Cloning Vectors

A

Plasmids
Bacteriophages: used to clone larger fragments
For much larger fragments: yeast artificial chromosomes, bacterial artificial chromosomes

26
Q

Cloning in Mammals

A
  1. To achieve gene over-expression or knock out to study the function of that gene
  2. For production of recombinant proteins in mammalian cells
  3. Gene therapy to treat human disease
27
Q

Bacteriophages

A
  • 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
28
Q

Viruses as Cloning Vectors

A
  1. Adenoviruses: will take DNA fragments up to 8kb. Induce inflammatory genes e.g. IFNg in transduced cells
  2. 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
  3. Adeno-associated viruses*: Inserts into host DNA at same position within human chromosome 19
  4. Retroviruses (e.g. Lentivirus)*: commonly used in gene therapy. Inserts at random positions → very stable integrants
29
Q

Lentivirus

A
  • 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
  • The 3 plasmids are transfected into a cell line to produce the viral vectors, which are collected in the supernatant
30
Q

Yeast Artificial Chromosomes

A
  • YAC libraries useful for cloning very large DNA fragments (> 1 Mb) cloning large genes & creating libraries of large overlapping clones e.g. for individual chromosomes
  • YACs are hybrids of bacterial plasmid DNA & yeast DNA. Components required for replication/segregation of natural yeast chromosomes combined with E. coli plasmid DNA
  • YACs are grown in S. cerevesiae & contain selectable markers. Yeast selectable markers allow growth of transformant on selective media lacking specific nutrients (non-transformants unable to grow)
  • Yeast strains used are auxotrophic i.e. unable to make a specific compound e.g. If mutant strain is transformed with YAC containing intact gene → compensation for inactive gene & transfected cell grows on media lacking nutrient
  • YACs are unstable and frequently lose parts of the DNA during propagation
31
Q

Genomic Library

A
  • A collection of the total genomic DNA from a single organism inserted into a vector
  • Used for sequencing and cloning applications
  • Not practical to recover rare sequences directly from isolated nuclear DNA due to large amount of extraneous DNA sequences
  • 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 – does not target a specific gene but seeks to clone all the genes at one time hoping that at least 1 recombinant clone will contain part of the gene of interest
  • Isolated DNA is only partially digested by the chosen restriction enzyme so that not every restriction site is cleaved in every DNA molecule
  • Even if the gene of interest contains a susceptible restriction site some intact genes might still be found in the digest
32
Q

cDNA Library

A
  • 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
  • Does not contain untranslated region, e.g. promoters, introns
  • Inserted into a vector
  • Represents the transcriptome of the cells/tissues
  • Used for screening high expression or functional important genes and the isolation of coding sequences of genes
33
Q

Preparation of genomic libraries

A

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

  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
34
Q

Screening by Colony Hybridization

A
  • Phage particles & extracellular viral genomic DNA transferred to nitrocellulose filter
  • Nitrocellulose filter is incubated with
    Radiolabelled (or biotinylated) probe for gene of interest
  • Autoradiograph will locate the clone with gene of interest
  • Clone can be isolated & inoculated into new host cells for further amplification
35
Q

Probes

A
  • Probe - cloned piece of DNA containing portion of sequence for which you are searching
  • 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
36
Q

Complementary DNA library

A
  • Multi-cellular organisms have specialization of individual cells e.g. liver cells, brain cells etc
  • Each cell contains the same complement of genes but in different cell types different sets of genes are switched on while others are silent
  • 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
37
Q

cDNA cloning step 1

A

Introduction

  • mRNA cannot be cloned directly but a DNA, a copy of the mRNA, can be cloned
  • This conversion is accomplished by the action of reverse transcriptase & DNA polymerase
  • Reverse transcriptase makes a single-stranded DNA copy of the mRNA
  • Second DNA strand is generated by DNA polymerase & double- stranded product is introduced into an appropriate plasmid or l vector
38
Q

cDNA cloning step 2

A

First strand synthesis

  • Key to cDNA cloning procedure is synthesis of cDNA from mRNA template by reverse transcriptase
  • mRNA obtained & purified from other RNAs by trizol extraction & column purification
  • Reverse transcriptase cannot initiate DNA synthesis without a primer
  • 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
39
Q

cDNA cloning step 3

A

RNA degradation and second strand synthesis

  • After mRNA copied → single-stranded DNA (“first strand”) the RNA is partially degraded with ribonuclease (RNase) H1. Degrades RNA strand of RNA/DNA hybrid
  • Remaining RNA fragments act as primers for DNA pol1 “second strand synthesis” which uses the first as the template
  • Result is double-stranded cDNA
40
Q

cDNA cloning step 4

A

Ligation, transformation, screening

  • Ligate cDNA into a vector
  • cDNAs have no sticky ends so ligate blunt ends or attach sticky ends (for more efficient ligation)
  • cDNA clones are representative of mRNA present in original preparation
  • cDNA library would contain a large proportion of clones representing insulin mRNA (other clones will also be present)
  • Identify clones by hybridization of specific probe
41
Q

Transgenic Mice

A
  • 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
42
Q

Transgenic Mice and Polio

A
  • 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 cannot 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 & other pathologies similar to human disease
43
Q

Knockout Mice

A
  1. genes targeted in ES cells through homologous recombination (inserts foreign DNA into genome)
  2. targeted ES cells selected and expanded
  3. targeted ES cells injected into early mouse embryo
  4. embryo implanted into uterus of pseudopregnant mouse
  5. chimeric mouse mated with normal mouse
    - gene inactivated by replacing it with a gene of interest (antibiotic resistance gene serving as selectable marker)
44
Q

Knock-In Mice

A
  • gene of interest not deleted

- additional function is added

45
Q

Transgenic Mice

A
  1. recombinant DNA injected to male pronucleus of fertilized oocyte
  2. injected oocyte into early mouse embryo
  3. embryo implanted into uterus of pseudopregnant mouse
46
Q

Transgenic vs Knockout Mice

A

transgenic: DNA injected into zygote without homologous recombination
knockout: DNA injected into ES cells with homologous recombination (sub-type of transgenesis)

47
Q

Transgenesis

A

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

48
Q

Gene Cloning Uses in Agriculture

A
  1. engineered crops
  2. cloning of genes in animals
  3. cloning of animals
49
Q

BT Corn

A
  • protects corn against corn borers laying eggs under the leaves
  • allows plants to express insecticides directly in the plant
  • Bacterium Bacillus thuringiensis (BT) has evolved defence mechanism to survive in the gut of insects by producing d-endotoxin, CryIA(b)
  • Accumulates as an inactive precursor in bacteria but after ingestion by insect protoxin 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
  • 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 & polyadenylation signal (required for production of mature mRNA for translation) from cauliflower mosiac virus
  • Introduced into maize embryos by mircroprojectile bombardment
50
Q

Golden Rice

A
  • helps Vit A deficiency that causes irreversible blindness

- rice engineered to express B-carotene precursor for vit A

51
Q

Gene Cloning in Medicine

A
  1. production of recombinant pharmaceuticals
  2. identification of disease causing genes
  3. gene therapy and cancer
52
Q

Factor VIII

A
  • Central role in blood clotting
  • Failure to synthesize factor VIII causes haemophilia
  • Until recently treat haemophilia by injection of purified factor VIII (can’t remove virus particles)
  • dimeric protein with 17 disulphide bonds
  • cloned as 2 subunits with each cDNA fragment ligated into an expression vector between promoter and polyadenylation signal
  • the plasmids are inserted into a hamster cell line to produce the factor
53
Q

Cystic Fibrosis

A
  • cystic fibrosis is an autosomal recessive genetic disorder
  • causes abnormal transport of chloride and sodium ions across epithelium
  • mutated gene for CFTR ion channel for chloride ions causing ion accumulation inside the cells and sticky mucus to build up outside the cells
54
Q

Nav1.7 Mutations

A
  • voltage gated sodium channel
  • single point mutation from TCA coding serine to TGA coding stop
  • causes complete inability to sense pain
  • knockout mice used to show that with this channel there is a loss of function mutation and a reduced response to mechanical stimuli
55
Q

Gene Therapy

A
  • Basic concept of gene therapy involves introducing gene → target cells to cure, prevent or slow down the progression of disease
  • Manipulation of cells removed from organism which are transfected & placed back e.g. stem cells from bone transfected with DNA used in the treatment of blood disorders
  • Cancer cell gene therapy → anti-sense RNA to silence oncogene
56
Q

Cystic Fibrosis gene therapy

A
  • Cystic fibrosis is a good candidate as it is caused by mutations in a single gene
  • A modified common cold virus was used as a delivery vehicle or vector to carry the normal CFTR gene to the cells in the airways of the lung
57
Q

Congenital Blindness gene therapy

A
  • One type of congenital blindness is caused by a mutation in the Retinal Pigment Epithelium-specific 65 kDa protein (RPE65)
  • RPE65 process 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
  • 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
58
Q

Forensic Science

A
  • 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

Molecular Biology (11 decks)

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