OVERVIEW
Biotechnology
- The use of microorganisms, cells or cell
components to make a product.
- Microbes used to make food, vaccines,
antibiotics and vitamins.
- Bacteria used to extract minerals in mining.
- Animal cells used to produce viral vaccines.
- Products made by cells were all naturally
made by cell until the 1980s.
One area of biotechnology is genetic engineering: |
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- The use of microorganisms,
plants (and sometimes animals) for the production of molecules they do
not normally make
- Involves inserting foreign
genes into cells
One area of genetic engineering involves Recombinant
DNA:
- DNA segments
are cut and joined in an environment outside a cell or organism. Under
appropriate conditions, a recombinant DNA (rDNA) molecule can enter a cell
and replicate there, either autonomously or after it has become integrated
into a cellular chromosome.
How to create recombinant DNA (short version):
1. Treat the DNA taken from both sources
with the same restriction endonuclease.
2. The restriction enzyme cuts both molecules
at the same site.
3. The ends of the cut have an overhanging
piece of single-stranded DNA called “sticky ends.”
4. These sticky ends can base pair with
any DNA molecule that contains the complementary sticky end.
5. Complementary sticky ends can pair with
each other when mixed.
6. DNA ligase is used to covalently link
the two strands into a molecule of recombinant DNA.
7. In order to be useful, the recombinant
DNA needs to be replicated many times (i.e. cloned). Cloning can be done
in vitro, via the Polymerase Chain Reaction (PCR), or in vivo (inside the
cell) using unicellular prokaryotes (e.g. E. coli), unicellular eukaryotes
(e.g. yeast), or mammalian tissue culture cells. |
RECOMBINANT DNA:
- Can be formed naturally
among related bacteria
- In the lab involves
joining of foreign DNA (genes) with bacterial DNA
- Expression of gene results
in production of protein
- Used to produce large
amounts of important proteins (e.g. insulin)
- Can also be used to
make thousands of copies (amplify) the inserted DNA
The DNA is "cut" into pieces using Restriction enzymes:
- Special class of DNA-cutting
enzymes (endonucleases) isolated from bacteria
- Protect bacteria by
hydrolyzing (thereby destroying) phage DNA during viral infections
- Bacterial DNA protected
by methylation (addition of methyl groups) of bases
- Recognizes and cuts
only one particular sequence of bases in DNA
- Cuts
sequence in the same way each time
- Recognize
4, 6 or 8 base pair sequences
- Often
make staggered cuts (which results in sticky ends)
- Two different pieces
of DNA cut with the same enzyme will have compatible sticky ends
- DNA can be spliced (recombined)
in
vitro (outside of the cell)
- DNA ligase used to link
the backbones of the DNA molecules together
Vectors
- Must be self-replicating
- Must be of a convenient size for manipulations
outside of cell
- Must be able to preserve itself from destruction
(circular or inserts in chromosome)
- Often contain selectable markers
- Plasmids
- One form of vector
- Are protected by circular
form
- Shuttle vectors are
capable of existing in several species
- Can be used to move
cloned DNA among different organisms
- Viral vectors
- Can accept larger pieces
of foreign DNA
- Infection of host cell
with virus will result in delivery of gene to host cell
- DNA may be inserted
in the chromosome of the host cell
Polymerase chain reaction (PCR)
- Used to amplify small samples of DNA
- Requires i) template DNA, ii) primers,
iii) nucleotides and iv) polymerase
- All reagents added to single tube and
placed in thermocycler
- Uses DNA from Thermus aquaticus (very
heat stable)
- Can only be used to amplify relatively
small segments of DNA (2-3 genes)
- Can be used in diagnostic work (detection
of pathogen DNA)
Inserting foreign DNA into cells
- Transformation - cells take up naked DNA
from the environment
- Many cells must be chemically treated
to become competent
- Electroporation - uses electrical current
to create pores in cell membrane
- DNA enters cell through pores
- Gene gun uses particles of tungsten or
gold coated with DNA
- Gun shoots particles into cells
- Some cells express introduced DNA as if
it were its own
- Microinjection uses glass pipette to inject
foreign DNA into cell
Obtaining DNA
- Gene libraries - DNA isolated from organism
and digested into fragments
- Fragments inserted into
vector
- Recombinant DNA introduced
into bacterial cells
- Results in collection
of clones containing all DNA fragments from organism
- Eukaryotic genes carry introns
- Translation of genes in bacteria would
result in garbage
- Use mRNA transcripts as source of gene
(already processed)
- Enzyme (reverse transcriptase) used to
synthesize complementary DNA (cDNA)
- mRNA enymatically digested away
Synthetic DNA
- Genes can be made in vitro using DNA synthesis
machine
- Only small pieces of DNA can be synthesized
- Need to link small pieces together to
form gene
- Requires knowledge of gene sequence
Making a gene product
- Early work done with E. coli as host for
recombinant DNA
- Genes expressed under control of E. coli
promoters
- Disadvantages
- E. coli contains endotoxin
(LPS) which causes fever and shock
- E. coli doesnÕt
secrete very well (cells need to be broken to obtain protein)
- Gram positive bacteria
(B. subtilis) often used for secreted proteins
- Yeast often used for expression of eukaryotic
genes
- Yeast used for secreted proteins
- Animal cells in culture often used to
express eukaryotic genes
- Also used as hosts to grow viruses- more
later
- Plant cells can be used to express genes
and to create genetically engineered plants
General molecular biology techniques utilized during recombinant
DNA include:
1. The study and/or alteration of gene expression
patterns
Gene expression is the process by which a gene's coded information is converted
into the structures present and operating in the cell. Expressed genes
include those that are transcribed into mRNA (messenger RNA) and then translated
into protein, and those that are transcribed into tRNA (transfer RNA) and
rRNA (ribosomal RNA). Gene expression can be studied using microarray analysis,
which is a method of visualizing the patterns of gene expression of thousands
of genes using fluorescence or radioactive hybridization.
2. Gene cloning
Gene cloning utilizing recombinant DNA technology is the process of manipulating
DNA to produce multiple copies of a single gene or segment of DNA.
3. DNA sequencing
DNA sequencing is a lab technique used to determine the sequence of nucleotide
bases in a molecule of DNA.
4. Creation of transgenic plants and animals
A transgenic plant or animal is one who has been genetically engineered,
and usually contains genetic material from at least one unrelated organism,
such as from a virus, other plant, or other animal.
Specific Applications
Medical
- Production of insulin and other hormones
(somatostatin, tissue-plasminogen activator)
- subunit vaccines - protein of pathogen
produced by harmless microorganism/ no chance of becoming infected
- Gene therapy - involves delivery of gene
to host cell using viral vector
- DNA integrates
into host cell chromosome; hopefully the gene is expressed and protein
is produced
Agricultural
- Ti plasmid of Agrobacterium tumefaciens
can integrate into plant chromosomes
- Insertion of foreign DNA into Ti plasmid
leads to insertion into chromosome
- Clones carrying foreign DNA produce genetically-altered
plants
- Anti-sense DNA technology- injected anti-sense
DNA binds to mRNA
- Protein not produced and RNA degraded
(used to preserve tomatoes)
- Increased nitrogen fixation in bacteria
- Possibility of producing plants containing
N-fixing genes
- Bovine growth hormone- produced recombinantly
and injected into cows
Scientific
- DNA sequencing - allows for determination
of entire genome of an organism
- using genetic code, protein sequences
can be determined
- Southern blotting - used for genetic screening
and DNA fingerprinting
Safety issues and ethics in genetic engineering
- labs must meet rigorous standards
- genes required for growth outside lab
often deleted
- recombinant microorganisms sometimes carry
suicide genes
- genetically engineered food may be toxic
- cross-pollination of weeds making them
herbicide resistant
- genetic screening of individuals
- technology only 30 years old ... where
will it lead?
Nucleotide
Activity
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