Biotechnology and Genomics Chapter 16. Biotechnology and Genomics 2 DNA Cloning: Gene Cloning is the...
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Transcript of Biotechnology and Genomics Chapter 16. Biotechnology and Genomics 2 DNA Cloning: Gene Cloning is the...
Biotechnology and Genomics
Chapter 16
2Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
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.
3Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Recombinant DNA Technology
Recombinant DNA (rDNA) contains DNA from two or more different sources, such as a human cell and a bacterial cellThis requires:
1. A vectorThis is a means to transfer foreign genetic material into a cell.
Plasmids (small accessory rings of DNA from bacteria) are common vectors
4Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Plasmids
There are many kinds of plasmidsR 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
5Biotechnology aBiotechnology and Genomicsnd GenomicsDNA 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
6Biotechnology aBiotechnology and Genomicsnd GenomicsDNA 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
7Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Restriction Enzymes
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.
8Biotechnology aBiotechnology and Genomicsnd Genomics
Restriction Enzyme Videos
Restriction Endonucleases
http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
9Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Restriction Enzymes
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.
10Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Restriction Enzymes
11Cloning a Human Gene
12Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Creating Custom Bacteria
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
13Cloning a Human Gene
14Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Creating Custom Bacteria
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
15Cloning a Human Gene
16Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Creating Custom Bacteria
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
17Cloning a Human Gene
18Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Creating Custom Bacteria
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
19Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Steps in Cloning a Gene
Steps in Cloning a Gene
http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf
20Cloning a Human Gene
21Biotechnology aBiotechnology and Genomicsnd GenomicsEarly Genetic
Engineering Experiment
Early Genetic Engineering Experiment
http://highered.mcgraw-hill.com/olc/dl/120078/bio38.swf
22Biotechnology aBiotechnology and Genomicsnd GenomicsDNA 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
23Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
cDNA Video
cDNA Video
http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf
24Biotechnology aBiotechnology and Genomicsnd GenomicsDNA 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
25Biotechnology aBiotechnology and Genomicsnd GenomicsDNA Cloning:
Polymerase Chain Reaction (PCR)Steps of PCR technique:
1. DNA to be copied is mixed with heat-resistant 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
26Biotechnology aBiotechnology and Genomicsnd GenomicsVideo
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction Video
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
27PCR
28Biotechnology aBiotechnology and Genomicsnd GenomicsApplications of PCR:
Analyzing DNA Segments
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
29Biotechnology aBiotechnology and Genomicsnd GenomicsGel Electrophoresis
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
30Biotechnology aBiotechnology and Genomicsnd GenomicsGel Electrophoresis
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 gelb. 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
31Biotechnology aBiotechnology and Genomicsnd GenomicsRestriction Length Fragment
Polymorphisms Video
Restriction Length Fragment Polymorphisms Video
http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf
32DNA Fingerprinting & Paternity
33Biotechnology aBiotechnology and Genomicsnd GenomicsGel Electrophoresis
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 .
34Biotechnology aBiotechnology and Genomicsnd GenomicsBiotechnology Products:
Transgenic Bacteria
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)
35Genetically Engineered Bacteria
36Biotechnology aBiotechnology and Genomicsnd GenomicsBiotechnology Products:
Transgenic Plants and Animals
Transgenic Plants have been created to produce:Pest resistant agricultural cropsHuman hormones
Transgenic AnimalsMany 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
37Biotechnology aBiotechnology and Genomicsnd GenomicsBiotechnology 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 fibrosisCancerBlood diseases, etc.
38Transgenic Mammals
39Biotechnology aBiotechnology and Genomicsnd Genomics
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.
40Biotechnology aBiotechnology and Genomicsnd Genomics
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
41Gene Therapy
42Biotechnology aBiotechnology and Genomicsnd Genomics
Gene Therapy
• In Vivo Gene Therapy
◊ Genes inserted in human body, via nasal sprays, viruses, or liposomes
Examples: - Cystic Fibrosis treatments
- Cancer therapies
43Biotechnology aBiotechnology and Genomicsnd Genomics
See Human Genome Project PowerPoint Presentation