Transcript Biotechnology and Genomics

Chapter 14
Biotechnology
and Genomics
DNA Cloning:
Biotechnology
and Genomics
Gene Cloning is the production of genetically
identical copies of DNA, cells, or organisms.
- Can be done to:
♦ Create many identical copies of the same gene
(gene cloning)
♦ Genetically modify organisms in a beneficial way
● When used to modify a human it is called gene
therapy.
● When used to modify another organism the
new organisms created are called transgenic
organisms.
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DNA Cloning:
Recombinant DNA Technology
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Recombinant DNA (rDNA) contains DNA from two
or more different sources, such as a human cell
and a bacterial cell
This requires:
1. A vector
 This is a means to transfer foreign genetic
material into a cell.
 Plasmids (small accessory rings of DNA
from bacteria) are common vectors
DNA Cloning:
Plasmids
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There are many kinds of plasmids
R plasmids carry genes for enzymes that
destroy antibiotics:
1. Thus, bacteria that carry these plasmids
and their genes are resistant to antibiotics
●This means they are not killed by the
antibiotic.
● This is a serious problem for humans as
more and more bacteria that cause
human diseases are becoming resistant
to our antibiotics
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Biotechnology
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DNA Cloning:
Recombinant DNA Technology
Recombinant DNA technology
 A set of techniques for combining genes
from different sources and then transferring
the resulting rDNA into cells.
1. Plasmids have been very useful in this
endeavor
2. Viruses have also been used for this
purpose
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DNA Cloning:
Recombinant DNA Technology
How to create recombinant DNA (rDNA)?
1. You must insert one organism’s DNA into the
vector DNA
2. To do this you need the following:
 A restriction enzyme – which cleaves, or
cuts, DNA
 A DNA ligase enzyme - seals DNA into the
DNA of the vector
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DNA Cloning:
Restriction Enzymes
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Restriction Enzymes
1. These are “cutting” tools for making rDNA
2. They are actually bacterial enzymes called
endonucleases.
 They are produced by bacteria to protect
themselves from invading viral DNA
 They work by finding and chopping out the
foreign DNA.
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Restriction Enzyme Videos
Restriction Endonucleases
http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
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DNA Cloning:
Restriction Enzymes
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How do restriction enzymes work?
1. They recognize short nucleotide sequences
in the DNA and cut at very specific points
2. They produce staggered cuts which are
called “sticky ends”.
 These single-stranded ends of DNA can
base-pair with any other piece of DNA
which was cut with the same restriction
enzyme.
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DNA Cloning:
Restriction Enzymes
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Cloning a Human Gene
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DNA Cloning:
Creating Custom Bacteria
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Steps to make custom bacteria:
1. Obtain 2 kinds of DNA:
a. Human DNA of interest
Example: gene for human insulin
b. Bacterial plasmid
This will act as a vector to get human DNA
into a bacterium
Cloning a Human Gene
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DNA Cloning:
Creating Custom Bacteria
Biotechnology
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2. Treat plasmid and human DNA with the
same restriction enzyme
a. Pick a restriction enzyme that:
• Cuts the plasmid in only ONE location
(just to open up the plasmid)
• Cuts the human DNA in just TWO spots
◊ Just in front of the gene for insulin
◊ Just behind of the gene for insulin
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Cloning a Human Gene
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DNA Cloning:
Creating Custom Bacteria
Biotechnology
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3. Mix the human DNA with the cut plasmid
a. The sticky ends of the plasmid should
base-pair with the complementary sticky
ends of the human DNA
b. Use DNA ligase to join the two DNA
molecules together with covalent bonds
c. The result is recombinant DNA
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Cloning a Human Gene
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DNA Cloning:
Creating Custom Bacteria
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4. Mix the recombinant DNA plasmid with
bacteria
a. Given the right conditions, the bacteria
take up the plasmid DNA by a process
known as transformation
5. The bacteria then reproduce asexually and
“clone” themselves.
a. All of these cloned bacteria will have the
gene for human insulin
◊ They will now produce insulin for us
DNA Cloning:
Steps in Cloning a Gene
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Steps in Cloning a Gene
http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf
Cloning a Human Gene
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Early Genetic
Engineering Experiment
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Early Genetic Engineering Experiment
http://highered.mcgraw-hill.com/olc/dl/120078/bio38.swf
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DNA Cloning:
How to get the gene for human insulin?
Gene given to the bacteria must NOT contain
introns. How do you get intron free DNA?
1. Obtain cells from tissues that produce a
lot of the desired protein
a. In this case, get pancreatic cells
2. Isolate the mRNA from these cells that
codes for insulin
3. Use the enzyme, reverse transcriptase, to
make DNA from the mRNA.
◊ This is called complementary DNA, cDNA
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DNA Cloning:
cDNA Video
cDNA Video
http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf
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DNA Cloning:
Polymerase Chain Reaction (PCR)
Technique to create copies of pieces of DNA
quickly in a test tube. It is said to amplify a
targeted sequence of DNA
Allows thousands of copies to be made of small
samples of DNA
Requires:
DNA polymerase
A supply of nucleotides for the new DNA strands
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DNA Cloning:
Polymerase Chain Reaction (PCR)
Steps of PCR technique:
1. DNA to be copied is mixed with heatresistant versions of DNA polymerase and
loose nucleotides
a. These were discovered in the bacterium,
Thermus aquaticus, which lives in hot
springs.
2. High heat (> 94oC) is applied to test tube
3. H-bonds break & DNA splits
4. Cool down test tube; DNA replicates
5. Repeat steps over and over
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Video
Polymerase Chain Reaction (PCR)
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Polymerase Chain Reaction Video
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
PCR
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Applications of PCR:
Analyzing DNA Segments
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DNA can be subjected to DNA fingerprinting
Treat DNA segment with restriction enzymes
-This creates a unique collection of different
fragments which differ from each other based
on their lengths. (RFLPs = restriction
fragment length polymorphisms)
-Gel electrophoresis separates the fragments
according to their charge/size
-Produces distinctive banding pattern
Gel Electrophoresis
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Gel electrophoresis is a method of physically
sorting macromolecules (DNA or proteins)
 Steps of procedure:
1. Samples of DNA are placed in wells at one
end of flat, rectangular gel
2. Gel is placed in an box with + and - electrodes
at each end
a. DNA, (-) charged, is placed at (-) end
b. Electric current pulls the DNA thru gel
towards the (+) electrode
Gel Electrophoresis
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3. As pieces of DNA move through the gel,
they will separate according to their size:
a. Smaller pieces move more quickly and
end up traveling further through the gel
b. Larger pieces will be bogged down in gel
and not move as far in the same amount
of time
4. You end up with “bands” in each lane of the
gel.
a. Each band represents a different size
DNA
Restriction Length Fragment
Polymorphisms Video
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Restriction Length Fragment Polymorphisms Video
http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf
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DNA Fingerprinting & Paternity
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Gel Electrophoresis
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5. If DNA from different people is run thru the
same gel, differences and similarities can
be studied
6. DNA fingerprinting is used to:
a. Measure number of repeats of short
sequences of DNA. People differ in the
number of these repeats.
b. Used in paternity suits, rape cases,
corpse ID, identification of viral infections,
identifying people with genetic disorders,
detection of cancer, identification of trade
in endangered species .
Biotechnology Products:
Transgenic Bacteria
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Transgenic organisms have had a foreign gene
inserted into their genetic make-up
Transgenic bacteria now produce:
Insulin, Human Growth Hormone, clotting factor
VIII, hepatitis B vaccine
Oil-Eating Bacteria
Promote plant health (Ice-minus strawberries)
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Genetically Engineered Bacteria
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Biotechnology Products:
Transgenic Plants and Animals
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Transgenic Plants have been created to produce:
Pest resistant agricultural crops
Human hormones
Transgenic Animals
Many types of animal eggs have taken up the
gene for bovine growth hormone (bGH)
-The procedure has been used to produce
larger fishes, cows, pigs, rabbits, and sheep
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Biotechnology Products:
Transgenic Plants and Animals
Gene Pharming:
-Use of transgenic farm animals to produce
pharmaceuticals
-Genes coding for therapeutic & diagnostic
proteins are incorporated into an animal’s
DNA
-The proteins appear in the animal’s milk
-Plans are to produce drugs to treat:
 Cystic fibrosis
 Cancer
 Blood diseases, etc.
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Transgenic Mammals
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Human Genome Project
Genome - All the genetic information of an
individual (or species)
Goals of Human Genome Project:
Determine the base pair sequence of human DNA
- Launched in 1990; completed a working draft in
2003
Construct a map showing sequence of genes on
specific chromosomes (approximately 25,000
genes code for proteins)
Other species sequences are being determined.
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Gene Therapy
The insertion of genetic material into human cells
for the treatment of a disorder
 Ex Vivo Gene Therapy
◊ Genes combined outside of body and then
placed inside
Examples:
- Children with Severe Combined
Immunodeficiency injected with modified
bone marrow stem cells
Gene Therapy
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Biotechnology
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Gene Therapy
• In Vivo Gene Therapy
◊ Genes inserted in human body, via nasal
sprays, viruses, or liposomes
Examples:
- Cystic Fibrosis treatments
- Cancer therapies
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See Human Genome
Project PowerPoint
Presentation
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