Biotechnology Definitions

Biotechnology Definitions

• Isoschizomers are a pair of restriction endonucleases with the ability to identify the same target sequence
• One of the enzymes can specify methylated and unmethylated target sequences
• Neoschizomer refers to an enzyme recognizing the same sequence as another enzyme, but cuts at different locations
• Isocaudomer refers to enzymes that recognize different sequences but produce the same sticky ends
• Vector is a DNA molecule capable of autonomously replicating in a host cell and integrating DNA fragments for cloning
• Cloning vectors are all vectors used for propagation of DNA inserts in a host
• Expression vectors are designed for gene expression by copying and turning genes into proteins
• Translational fusion refers to the expression of fusion proteins comprising amino acids encoded by a sequence in the vector and the DNA insert
• Transcriptional fusion is the synthesis of pure proteins encoded exclusively by DNA inserts
• Plasmids are extrachromosomal DNA capable of independent replication, primarily found in bacteria
• Patent is a government-granted right excluding others from commercial use of an invention for a specified period
• Copyright is the protection of intellectual properties that aren't patentable

Steps in Gene Cloning

• Production and isolation of DNA fragments
• Insertion of isolated gene in a vector to obtain rDNA
• Introduction of rDNA into a host cell, usually E. coli
• Selection and identification of transformed host cells containing a gene of interest
• Multiplication or expression of the introduced gene in the host

Types of Restriction Endonucleases (REs)

• REs are classified based on structure, function, and recognition
• REs cut DNA at specific sequences

Type I REs

• Posses a complex function
• Requires ATP
• Involved in both restriction and modification of DNA
• Cutting occurs randomly, far from the recognition site
• EcoKI is an example

Type II REs

• Are most commonly used
• Does not require ATP
• Requires Mg2+ ions
• Cuts DNA at specific sequences
• Enzyme function is independent of methylase activity
• Cutting occurs at or near the recognition site
• Examples of Type II REs include EcoRI and BamHI

Type III REs

• Has function intermediate between type I and type II
• Cleaves at a short distance from the recognition site
• Requires ATP
• Also has a modification (methylation) function
• Cleaves a few bases away from the recognition site
• EcoP151 is an example

Type IV REs

• Recognizes methylated DNA
• Cuts methylated but not unmethylated DNA
• Mspl is an example

Type V REs

• Targets specific sequences based on RNA guides
• Requires guide RNA to direct them to the target DNA sequence
• Cas9 is an example

Summary of RE Types

• Type I modifies and cuts DNA randomly and performs complex functions
• Type II cuts at or near recognition site and is the most commonly used in labs
• Type III cuts away from the recognition site and involves methylation
• Type IV cuts methylated DNA
• Type V is guided by RNA and includes CRISPR-associated enzymes (like Cas9)

Commonly Used Vectors

• Plasmids
• Bacteriophages
• Cosmids
• Phasmids
• Shuttle vectors
• Artificial chromosomes (BAC/YAC)
• Phagemids

Specific Vectors

• pBR322 is the most widely used plasmid, stands for Plasma Bolivar Rodriguez
• pUC18/19 stands for Plasmid University California, and is a derivative of pBR322

Genetic Assays

• DNA fingerprinting is the description of specific alleles present at polymorphic loci in the genome. Discovered by Alec Jefferys in 1986
• Sequencing determines the order in which nucleotides are arranged in DNA
• BLAST (Basic Local Alignment Search Tool) identifies potential homologues for a given sequence

Restriction Endonucleases

• They're also called molecular scissors
• Type I was first reported by Arber and Meselson in 1960
• Type II was reported in 1970
• Endonucleases produce internal cuts called 'cleavage'
• Endonucleases recognize and cleave exogenous DNA in cells, acting as a defense mechanism against bacteriophages
• REs recognize specific sequences of nucleotides 4-8 base pairs long at a restriction site or recognition site
• Recognition sites are usually palindromic sequences (Type II)

Mechanism of Action

• Enzymes bind to DNA at recognition sites
• Enzymes active binding site is specifically shaped to interact with the bases of the recognition sequence
• After binding, the enzyme cuts phosphodiester bonds between nucleotides in DNA and is dependent on restriction enzyme to cut.
• After cleavage, there are 2 fragments of DNA remaining
• Cuts can be staggered/sticky/cohesive where 2 protruding ends readily pair with each other or even/blunt/flush
• Bacteria use methylation to modify their own restriction sites, preventing self-cleavage
• These are important for biotechnology in gene cloning

Properties of a Good Vector

• Should have an origin of DNA replication
• Should be able to replicate autonomously
• Ideally should be <10KB in size
• Easy to isolate and purify
• Easily introduced into host cells
• Should have a suitable marker
• Has the ability to integrate either itself or DNA in the genome of host cells
• Cells transformed with good vectors should be identifiable or selectable
• Should contain unique target sites for as many RE's without disrupting the essential functions
• Expression of DNA if required then vector should have suitable control elements such as promoter, operator, and RBS (ribosome binding sites)

Properties of Good Host

• Easy to transform
• Supports replication
• Free from elements that interfere with replication of rDNA
• Lacks active REs (e.g., E. coli K12 substrain HB101)
• Should not have methylases
• Deficient in normal recombinant function
• Bacteria are the host of choice for DNA cloning
• E. coli K12 is most commonly used

Transformation Methods

• Transformation involves the absorption of soluble naked DNA that is present in the micro-environment by a recipient cell including further inclusion in the chromosome

3 Methods of Transformation are:

• Calcium Chloride Method
• E. coli cells and plasmid DNA interact productively in an environment of Ca2+, low temperature, and subsequent heat shock
• CaCl2 affects cell wall
• Actual uptake of DNA is stimulated by brief heat shock
• Electroporation
• Zimmerman and Vieken - 1983 Rapid and simple technique for introducing a cloned gene It can be used for microbes, plant cells and animal cells
• High voltage electric pulses induce cell's plasma membrane to fuse and allows for cells to take up exogenous DNA from the suspending solution
• Proportion of cells become stably transformed
• Factors affecting efficiency of electroporation: Temperature Electrical parameters (voltage, resistance and capacitance) Topological forms of DNA Host factors (genetic background, growth conditions)
• Gene gun
• Device that fires DNA into target cells
• DNA is coated onto microscopic beads of gold/tungsten
• Beads are attached to the end of a plastic bullet
• Bullets are loaded into the firing chamber of the gene gun Explosive force applied to fire bullet towards the target cells that lie beyond the end of the barrel
• DNA coated beads continue towards the target cells
• Some beads pass through the cell wall and into the cytoplasm of the target cells

Methods of Transfection

• Transfection is the process of inserting double-stranded DNA/RNA into mammalian cells enabling expression or protein production using the cell's machinery Animal cells can perform post-translational modification
• Cell cultures are used on a commercial scale.
• There are 6 methods

DNA/Calcium Phosphate Co-precipitate Method

• HEPES buffered saline solution containing phosphate ions is combined with calcium chloride solution containing DNA to be transfected
• When the 2 are combined, a fine precipitate of positively charged calcium and negatively charged phosphate will form
• This binds the DNA to be transfected on its surface
• Suspension of precipitate is then added to the cells to be transfected
• Precipitate must be formed freshly
• Small granules of calcium phosphate associated DNA are endocytosed and transferred into the nucleus where the complex is expressed
• It's only suitable for cells growing in monolayers since precipitate must coat the cells
• Low proportion of cells take up DNA (1-2%)

Chemical Transfection

• Some cell lines are adversely affected by co-precipitation
• DEAE dextran promotes interactions between DNA and endocytic machinery
• Cationic polymers bind negatively charged DNA
• Polybrene is more efficient (instead of DEAE dextran)

Lipofection

• Liposomes are cationic/neutral liquid solutions
• Liposomes entrap DNA which is then fused with the cell membrane of the host
• Mechanism- Liposomes can spontaneously form stable complexes with DNA and Interact with cell membrane resulting in endocytosis
• Advantageous because it's Suitable for transient and stable transformation and Sufficient to be used YACs and large DNA fragments

Electroporation

• Generates transient nanometer-sized pores in the cell membrane, using electricity
• DNA enters cell through nanopores and is transported to cell nucleus

Direct transfer method

• DNA is injected to cell nucleus
• Allows a guaranteed success on target cell, but it's disadvantagous since it can only be applied to a few experitments

Peptides

• Can bind to and condense DNA, E.g., Serine-Proline-Lysine-Lysine (tetrapeptides)
• Positively charged lysine counters negative charge of phosphate backbone of DNA which allows DNA molecules to pack closely

Polymerase Chain Reaction (PCR)

• PCR amplifies a single copy or a few copies of DNA across generating a large number of copies of the sequence
• PCR targets and amplifies regions of DNA strand
• This test was invested in 1983 by Kary Banks Mullis
• Polymerase - only enzyme used in this reaction is DNA polymerase Products of the first reaction become substrates of the following one

Components of Reaction

• Target DNA (DNA sequence to be amplified)
• Pair of primers (Oligonucleotides that define the sequence to be amplified)
• dNTPs (DNA building blocks)
• Buffer solution (Maintains pH and the ionic strenth of teh reaction soltuion)
• Bivalent cations (Cofactor of the enzyme)
• Thermostable DNA polymease (Enzyme that catalyses the reaction)
• NFW (Nuclease free water)

Primers

• A short nucleic acid startsDNA synthesis
• Optimal length: 18-30 nucleotides
• Primers should not be comlimentary at the 3' ends
• Primers should contain 40-60% GC countent

Taq DNA polymerase

• Replicates DNA and is first discovered from Thermus aquaticus
• Enzyme works at 74 degrees celsius
• The enzyme is a 5'-3' polymerase activity and 5'-3' exonuclease activity
• The processivity, fidelity, and persistence of the enzyme is important

dNTPs

• They are building blocks
• Stability during repeated cycles of PCR is 50% after 50 cycles
• Concentration between 20-200 micromoles

Magnesium Chloride

• Specificity of PCR is dependent on its concentration
• This divalent cation is required for some Type II enzymes

Potassium Chloride

• Helps in elongation by reducing repulsion charges on the DNA strands

Steps of PCR

• Denaturation (94-98 degrees celsius) breaks hydrogen bonds between complementary bonds
• Annealing (50-65 degrees celsius) allows Primers to bind to single stranded DNA
• Extention (72 degrees celsius) synthesises a new strand

Variants of PCR

• Real time PCR (QPCR) allows quantification of specific NA
• Multiplex PCR allows amplification of multiple target sequence at once
• RT PCR converts RNA to DNA
• Nested PCR Involves sets of primers for 2 runs

Applications of PCR

• Molecular identification, DNA figerprinting, organisms etc.
• Sequencing, binformatics, genomic cloning, etc.
• Genetic engineering, site directed mutagenesis, genetic expression studies, etc.

BACS vs YACs

• A BAC is a Bacterial artificial chromosome, it's E. coli, and is derived from a bacterial F factor
• A YAC is a Yeast artificial chromosome, it's Saccharomyces cerevisiae, and it is derived from yeast chromosomes

Types of Fungi Classification

• Pathogenic fungi can be classified in 5 ways: Macroscopic morphology, Microscopic morphology, Type of reproduction, Taxonomy, and Mycosis

Macroscopic morphology

• Monomorphic yeasts multiply through budding of blastospores and resemble bacterial cultures
• Candida albicans is an example of a monomorphic yeast
• Monomorphic molds multiply by germ tubes from spores
• Aspergillus and Penicillium are example of of a monomorphic mold
• Dimorphic fungi can exist in both yeast and molds

Microscopic morphology

• There are 3 features, Hyphae Type, Type of Spores, and Spore bearing structure
• Septate hyphae has walls
• Aseptate has the absense of walls
• Pseudohyphae are attached elongated budding

Spores

• Asexual or Vegetative spores produce Macroconidia, Microconidia, Chlamydospores, Arthrospores, Blastospores, Sporangiospores
• Sexual produce Ascospores, and Basidospores, or Zygospores

Type of Spore Bearing Structures

• Conidiophore is like a branch of mycelium with Conidia
• Sporangiophore is bearing sporangium with the columella

Reproduction

• Perfect only reproduce sexually, and are exemplified my saccharonyces cerevisiae.
• Imperfect reproduce asexually and form a deuyeromcetes

Taxonomy

• Funi used to be classes as belonging to the phylum but were made their own kingdom
• They are classifed under the phylum, Class Ascomycetes, Class Basidiomycetes, and Class Deutomycetes

Mycosis

• This is when class is given to tissue being invaded (superficial, cutaneous, subcutaneous, systemic)

Ringoworms

• These are superficial infections of keratinized tissues.
• It's known as dermatophytosis or dermatomycosis
• It is a relatively rare infection in sheep and more rare in goats
• Transmission can be direct or indirect

Fungi (Ringworm)

• Trichophyton, Microsporum, and Epidermophyton are causative agents of
• Symptoms include Ring shaped lesions on the skin
• Diagnostic
• Examine lesions in dark room using UV light rays of 3660A (Wood's Lamp)
• Hair will produce yellowish green fluorescence

Aspergillosis

• This fungus cause Granulomatous, necrotizing and cavitory diseases
• It's found in the lungs
• Birds most commonly get affected as it's derived from poultry

The etiological agent of Aspergillosis

• Aspergillus fumigatus causes aspergillosis, along with A. nidulans or A. flavus
• There are both a cute and chronic symptoms that may occure when the bird gets it's
• Diagnosis: Take colonies, do a test, and see it under a microscope
• This test can be used to find if an animal has aspergillosis

Mycotic

• inflammation of mammary gland
• Is characterized by changes in milk secretion of the affected quarter/quarters
• This milk becomes unfit for human consumption Mycotic etiology
• Ainsworth and Austwick have described 26 species of fungi associated with mastitis
• Epidemiology, source of infection, and source of infection are common factors

Mycotic Abortions

• these abortions are common for goats
• It's associated with hay or straw
• it's caused by:Aspergillus fumigatus, A. flavus, A. nidulans,A. niger

Pathogenesis

• Inhilation, Ingetions,
• Causes fungal placentitis, resulting in death
• Based on hyphae, you diagnostic it

Mycotoxicosis

• This occurs upon injection
• Known as turkey x
• Can get it through the skin or injection

Pathogens

• High conc immediate toxin level
• Low conc are carcingenic