67 terms

Biotechnology for the 21st Century

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Clone
a molecule, cell, or organism that was produced from another single entity
Restriction Enzymes
DNA cutting enzymes, Primarily found in bacteria
Plasmid DNA Vectors
circular form of self-replicating DNA, can be manipulated to carry and clone other pieces of DNA
Palindromes
Recognition sequences where restriction enzymes attach and slice
Plasmid DNA
Most common, small circular pieces of DNA found primarily in bacteria, Can coexist with other types or are mutually exclusive depending on their "ori" sites
Types of Vectors
Bacterial plasmid vectors
Bacteriophage vectors
Cosmid vectors
Expression vectors
Bacterial Artificial Chromosomes (BAC)
Yeast Artificial Chromosomes (YAC)
Shuttle vectors
Ti vectors
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Features of a Good Vector
Size
Origin of replication (ori)
Multiple cloning site (MCS)
Selectable marker genes
RNA polymerase promoter sequences
DNA sequencing primers
Selection
a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria, like antibiotic or blue-white
DNA Libraries
Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host
Screened to pick out different genes of interest
Genomic Libraries
Chromosomal DNA from the tissue of interest is isolated and digested with restriction enzyme
Vector is digested with same enzyme and DNA ligase is used to ligate genomic DNA fragments and vector DNA
Recombinant vectors are used to transform bacteria
cDNA Libraries
mRNA from tissue of interest is isolated
Converted to a double-stranded DNA by using the enzyme reverse transcriptase
Called complementary DNA (cDNA) because it is an exact copy of the mRNA
mRNA is degraded
DNA polymerase used to create the second strand of DNA
Short linker sequences are added to the end of the cDNA
Contain restriction enzyme recognition sites
Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors
Vectors used to transform bacteria
Colony Hybridization
Bacterial colonies containing recombinant DNA are grown on an agar plate
Nylon or nitrocellulose filter is placed over the plate and some of the bacterial colonies stick to the filter at the exact location they were on the plate
Treat filter with alkaline solution to lyse the cells and denature the DNA
Denatured DNA binds to filter as single-stranded DNA
Filter is incubated with a probe
DNA fragment that is complementary to the gene of interest
Labeled with P32 or Fluorescent tag for easy detection
Probe binds by hydrogen bonding to complementary sequences on the filter
Polymerase Chain Reaction
Denaturation (94-960C)
Annealing (hybridization) (50 -650C)
Extension (elongation) (70 -750C)
DNA Sequencing
Chain termination sequencing (Sanger method), or computer automated sequencing
Fluorescence in situ hybridization (FISH)
Chromosomes are isolated from cells and spread out on glass slide
cDNA probe for gene of interest is labeled with fluorescent nucleotides and incubated with slides
Probe will hybridize with complementary sequences on slide
Slide is washed and exposed to fluorescent light
Wherever probe bound, it is illuminated to indicate the presence of that gene
Southern Blotting
Digest chromosomal DNA into small fragments with restriction enzymes Fragments are separated by agarose gel electrophoresis
Gel is treated with alkaline solution to denature the DNA
Fragments are transferred onto a nylon or nitrocellulose filter (called blotting)
Filter (blot) is incubated with a probe and exposed to film by autoradiography
Number of bands on film represents gene copy number
Northern Blotting
Basic method is similar to Southern blotting
RNA is isolated from a tissue of interest, separated by gel electrophoresis, blotted onto a membrane, and hybridized to a probe
Presence/absence/intensity of a band is conclusive
Reverse transcription PCR (RT-PCR)
Reverse transcription of mRNA is performed - converted into double-stranded cDNA
cDNA is then amplified with a set of primers specific for the gene of interest
Products electrophoresed on agarose gel
In situ hybridization
Used to determine the cell type that is expressing the mRNA
Tissue of interest is preserved in a fixative solution and embedded in a wax-like substance
Tissue can be sliced into very thin sections attached to microscope slides
Slides are incubated with a probe to the gene of interest
Probe hybridizes with mRNA in cells
Probe is detected
Microarrays
Single-stranded DNA molecules are attached onto a slide using a robotic arrayer fitted with tiny pins
Can have over 10,000 spots of DNA
Extract mRNA from tissue of interest, tag it with fluorescent dye, and incubate overnight with the slide
mRNA will hybridize to spots on the microarray that have complimentary DNA sequences
Slide is scanned with a laser that causes the spots to fluoresce
Genomics
cloning, sequencing, and analyzing entire genomes
Shotgun sequencing
The entire genome is cloned and sequenced
Produces thousands of fragments to be sequenced
Bioinformatics
An interdisciplinary field that applies computer science and information technology to promote an understanding of biological processes
The Human Genome Project
Started in 1990 by the U.S. Department of Energy
International collaborative effort to identify all human genes and to sequence all the base pairs of the 23 human chromosomes
Celera Genomics
Proteins
large molecules that are required for the structure, function, and regulation of living cells
Protein Structure
Primary structure is the sequence in which amino acids are linked together
Secondary structure occurs when chains of amino acids fold or twist at specific points (H-bonding)
Alpha helices and beta sheets
Tertiary structures are formed when secondary structures combine and are bound together covalently
Quaternary structures are unique, globular, three-dimensional complexes built of several polypeptides
Glycosylation
post-translational modification wherein carbohydrate units are added to specific locations on proteins
Upstream processing
the actual expression of the protein in the cell
Downstream processing
involves purification of the protein and verification of the function; a stable means of preserving the protein is also required
Chromatography
- allows the sorting of proteins based on size or by how they cling to or dissolve in various substances
Size exclusion chromatography
uses gel beads with pores
Larger proteins move quickly around the beads and smaller proteins slip through the pores and therefore move more slowly through the beads
Ion exchange chromatography
relies on the charge of the protein
Resin is charged
Opposite charged proteins will stick to resin beads
Can be eluted by changing the charge with salts of increasing concentration
Affinity chromatography
relies on the ability of proteins to bind specifically and reversibly to uniquely shaped compounds called ligands
Mass spectrometry
highly sensitive method used to detect trace elements
Used to indicate the size and identity of most protein fragments
High-Performance liquid chromatography
uses high pressure to force the extract through the column in a shorter time
Iso-electric focusing
QC to identify two similar proteins that are difficult to separate by any other means
Each protein has a specific number of charged amino acids on its surface in specific places
Creates a unique electric signature known as its iso-electric point (IEP) where charges on the protein match the pH of the solution
Lyophilization
Protein, usually a liquid product, is first frozen
A vacuum is used to hasten the evaporation of water from the fluid
Will maintain protein structure and can be stored at room temperature for long periods of time
Proteomics
A new scientific discipline dedicated to understanding the complex relationship of disease and protein expression, Uses protein microarrays to test variation in protein expression between healthy and disease states
Microbes
tiny organisms that are too small to be seen individually by the naked eye and must be viewed with the help of a microscope
peptidoglycan
What the bacterial cell walls are made of
Bacterial shapes
Cocci, Bacili, Spiral
Bioluminescence
method of producing light used by marine organisms
Antigens
foreign substances that stimulate an immune response
B cells
with the help of T cells, recognize and bind to the antigen, develop to form plasma cells that produce antibodies
Vaccines
parts of a pathogen or whole organisms that can be given to humans or animals by mouth or by injection to stimulate the immune system against infection by those pathogens
Subunit vaccines
are made by injecting portions of viral or bacterial structures (Hep B)
Attenuated vaccines
use live bacteria or viruses that have been weakened through aging or by altering their growth conditions to prevent replication (MMR)
Inactivated (killed) vaccines
are made by killing the pathogen and using the dead or inactivated microorganism for the vaccine (Salk polio)
DNA based vaccines
1st used in 2005, in horses against West Niles virus
Plant Transgenesis
transferring genes to plants directly
Development of plant vaccines, plants that produce their own pesticides and are resistant to herbicides
Protoplast fusion
the fusion of two protoplast cells from different species
Protoplast cell is a callus cell whose cell wall has been dissolved by the enzyme cellulase
Fusion of the two protoplast cells creates a cell that can grow into a hybrid plant
Examples include broccoflower (broccoli + Cauliflower), Triticale (wheat - triticum + rye - secale)
Leaf fragment technique
Small discs are cut from leaf
Cultured in a medium containing genetically modified Agrobacter (Agrobacterium tumefaciens)
A soil bacterium that infects plants
Bacterium contains a plasmid, the TI plasmid, that can be genetically modified
DNA from the TI plasmid integrates with DNA of the host cell
Leaf discs are treated with plant hormones to stimulate shoot and root development
Gene Guns
Used to blast tiny metal beads coated with DNA into an embryonic plant cells,
Aimed at the nucleus or the chloroplast
Use marker genes to distinguish genetically transformed cells
Antibiotic resistance
Technique is useful in plants that are resistant to Agrobacter
Chloroplast engineering
DNA in chloroplast can accept several new genes at once
High percentage of genes will remain active
DNA in chloroplast is completely separate from DNA released in pollen - no chance that transformed genes will be carried on wind to distant crops
Antisense technology
Process of inserting a complementary copy of a gene into a cell
Gene encodes an mRNA molecule called an antisense molecule
Antisense molecule binds to normal mRNA (sense molecule) and inactivates it
Example is Flavr Savr tomato (1994 - 1st GMF approved by FDA)
homologs
Many genes in different species have been shown to be similar to human genes based on DNA sequence
orthologs
In different species
paralogs
In the same organism - separated due to gene-duplication
Biomarkers
typically proteins produced by diseased tissue or proteins whose production is increased when a tissue is diseased
Allele-specific oligonucleotide analysis (ASO)
allows for the detection of single nucleotide changes even if the mutation does not change a restriction site
DNA microarrays
glass microscope slides spotted with thousands of genes
Can be used to screen a patient for a pattern of genes that might be expressed in a particular disease condition
Microspheres
- tiny particles that can be filled with drugs (=liposomes)
Made from materials that closely resemble lipids found in cell membranes
Mist sprayed in the nose to treat lung cancer and other respiratory illnesses; anticancer drugs; anesthetics for pain management
Oncogenes
genes involved in the growth of cancer cells
Tumor suppressor genes
produce proteins that keep cancer formation in check
Ex vivo gene therapy
Cells are removed from the patient, treated with techniques similar to transformation, and then reintroduced to the person
In vivo gene therapy
Introducing genes directly into tissues and organs in the body
Challenge is delivering genes only to intended tissues and not tissues throughout the body
RNA or gene silencing
Promising way to turn off disease genes
Used successfully in cell culture, but has yet to live up to its promise as a treatment for disease
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