Biotechnology and Genomics Chapter 16. Biotechnology and Genomics 2 DNA Cloning: Gene Cloning is the production of genetically identical copies of DNA,

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.


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


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



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



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


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




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.


Restriction Enzymes

11Cloning a Human Gene


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




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




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




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


Steps in Cloning a Gene

Steps in Cloning a Gene

http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf



21Biotechnology aBiotechnology and Genomicsnd GenomicsEarly Genetic

Engineering Experiment

Early Genetic Engineering Experiment




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


cDNA Video

cDNA Video

http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf




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


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



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


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



32DNA Fingerprinting & Paternity


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


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


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


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.


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


Gene Therapy

• In Vivo Gene Therapy

◊ Genes inserted in human body, via nasal sprays, viruses, or liposomes

Examples: - Cystic Fibrosis treatments

- Cancer therapies


See Human Genome Project PowerPoint Presentation

Documents

Biotechnology and Genomics Chapter 16. Biotechnology and Genomics 2 DNA Cloning: Gene Cloning is the production of genetically identical copies of DNA,