Biotechnology-the application of biological principles, organisms, and products to practical purposes; applied biological science

Molecular biology-the science dealing with DNA and protein synthesis of living organisms; the study of the storage and flow of information within a cell

Genetic engineering-manipulating genetic information in vitro

I-PCR

II-Making and using Recombinant DNA

A. restriction enzymes
B. vectors
C. steps

III-DNA Fingerprinting

A. RFLPs
B. Electrophoresis
C. Legal Applications
D. Other Applications

IV-DNA Sequencing

V-Probes

VI-Biotechnology Applications

A. Genetically engineered proteins

B. Genetically engineered vaccines

C. Gene Therapy

D. Genetically Modified Crops

E. Genetically Modified Animals

F. Human Genome Project

G. Current projects

 

I-PCR – Polymerase Chain Reaction

Steps

1. DNA is heated, -> single strands = template
2. nucleotides, enzymes (including polymerase), template & primers mixed together. Primers are a short, single strand of DNA that functions as a starting point for the polymerase chain reaction. Primers are chosen to amplify the desired section of DNA.
3. mixture cooled
4. new strand of DNA builds onto template (original strand), -> 2 double stranded DNA molecules
5. process repeated, -> 4 double strands, then 8, etc.

II-Making & Using Recombinant DNA
Cloning in genetic engineering – the linking of a specific gene or DNA fragment to a replicable DNA molecule, such as a plasmid.

A. Restriction Enzymes

Enzymes that cut DNA at a certain location. Location defined by sequence of bases

After restriction enzyme recognizes its specific sequence, will cut both DNA strands.

May cut straight across, -> blunt ends
OR

may cut one strand, split next few H bonds, cut other strand, -> sticky ends. "Sticky end" has unpaired bases.

B. Vectors
Vector = a DNA molecule, capable of replication, into which a gene or DNA segment is inserted by recombinant DNA techniques; a cloning vehicle
Plasmid – circular piece of DNA from bacteria that easily accepts another piece of DNA. After inserting the plasmid into bacteria, the bacteria will replicate new piece of DNA every time it duplicates the plasmid and its own.
Other systems accommodate larger pieces of DNA than plasmid can

C. Steps

1. Cut out desired piece of DNA with restriction enzyme
2. Use same restriction enzyme to cut vector. Leaves identical ends because restriction enzyme cuts same sequence in DNA from both sources.
3. If single stranded sticky ends, complementary single strands will associate by forming H bonds. (base pairing)
4. New piece of DNA has sequences from both sources = recombinant DNA – A DNA molecule composed of one or more segments from other DNA molecules
5. Insert recombinant DNA into cell that will make copies of recombinant DNA every time it makes copies of its own DNA.

III-DNA Fingerprinting

A. RFLPs = restriction fragment length polymorphisms
Restriction fragment = piece of chromosome cut by restriction enzyme
Polymorphism = many forms
RFLP = genetic variation in a population associated with the size of restriction fragments. The polymorphism results from the positions of restriction site flanking the probe.

Restriction enzymes cut different length pieces because:

1. restriction site may or may not be present

OR
2. variable length piece of DNA between restriction sites - due to many copies of the same short DNA sequences (tandem repeats), positioned one after the other along the length of a chromosome

Tandem Repeats = many copies of a repeated nucleotide sequence
Alleles stained for identification.
Variations are heritable & can be used as genetic markers

B. Electrophoresis

Gel is like agar (gelatin) – has holes of certain size that act as sieve.
Smaller pieces of DNA will go through gel easier and the larger ones will have a harder time squeezing through, -> slower.

Electrophoresis is the addition of an electrical current to the gel. The pieces of DNA have an electrical charge and are pulled through the gel by the electrical current. DNA has negative charge and will be pulled toward the positive end. Size (length) will determine how far the piece of DNA travels.

Pattern produced by electrophoresis = DNA Fingerprint

C. Forensic Application of DNA fingerprinting

Used in court cases as evidence
Know occurrence in population for each allele.
Multiply statistical chance of each allele by chance of next allele -> chance of it being same person.

This evidence submitted with others for final decision.
DNA fingerprint subject to same errors as other evidence.

D. Other applications

Family relatedness – paternity, immigration
Clues to inherited diseases
Evolution

IV-DNA Sequencing

Learn order of nucleotides in a gene and/or chromosome.
Analyzes DNA one base at a time, in sequence.

Does not tell function.

V-Probes

labeled sequence of single stranded DNA that will find to the gene of interest, if present

label can be radioactive or colored

Microarrays - a set of known single stranded, short, known genes attached to a solid surface. Unknown single stranded DNA sequences are added. If they bind, via complementary base pairing, they can be detected and identified by their position on the microarray.

VI-Biotechnology Applications

A. Genetically engineered proteins

Human insulin

Human growth hormone

Interferons

B. Genetically engineered vaccines - insert non-disease causing part of bacterial or viral DNA into potential host to cause host to produce disease-like proteins so that host will become immune to disease

C. Gene Therapy - insert correct copy of DNA into human so that it will make functional protein to compensate for disease causing protein. Examples: SCID (severe combined immunodeficiency) & cystic fibrosis

D. Genetically Modified Crops
Add gene to make crop

More productive
Tolerate harsh conditions (little water, salt in soil, cold climate, etc.)
Food supplement – Vitamin A rice
Natural pesticide – Ex: have added bacterial gene (Bt) to some crops so that bacterial insecticide will kill pest.
Advantage – much less chemical pesticides used – environmentally friendly, less expense. (No danger to non-insect species.)
Disadvantage – may harm insects besides crop pests.
Argument that insects will become resistant to Bt applies to any pesticide
Some countries require human food to be labeled if genetically modified, some don’t.

E. Genetically Modified Animals

Technique

Applications

a. Transgenic Animals as Human Disease Models

Easier to do research on and experiment with treatments on animals than humans.
Have been able to insert genes that -> some diseases into laboratory animals.
Can find cause, try treatments & doses in animals before doing human clinical trials.

b. Transgenic Animals with Enhanced Commercial Benefits.

more meat or faster growth

bovine Somatotropin (bST) -> more milk production

animals that produce products like like spider silk in their milk for human use

F. Human Genome Project
DNA sequence of 99.9% of the human genome mapped

Took DNA from 100 volunteers, only used some of them so no one knows if their was used or not

Publicly available DNA sequence of human genome allows research to find relationship between genes and their function, such as disease, growth, development , etc.

G. Current projects

Genomics -the study of genomes

Sequence DNA of other organisms to find similarities, differences
Find function of sequences

Proteomics - study of the structure, function and interaction of cellular proteins

Bioinformatics - use of computers to catalogue and compare genomic & proteomic information. Building, searching and applying databases.