Genetic Engineering

Chapter 13•Recombinant DNA

•Transformation•Biotechnology

•Gel Electrophoresis•PCR

Biotechnology- Any technological application that uses biological systems to make or modify products or processes

Genetic Engineering• Cohen and Boyer

constructed a creature that was part bacterium and part frog

• Vacanti grew an ear on a mouse

• How did they do this?

Involves Recombinant DNA• DNA that contains DNA segments from 2

separate organisms• Restriction enzymes are used to cut DNA

molecules only in specific places– Cut at a specific sequence of bases called the

recognition sequence– For example, Hind III enzyme recognizes and cuts

DNA at 5’AAGCTT3’– Break bonds between the 3’ hydroxyl group of 1

nucleotide and the 5’ phophate group of the next nucleotide

• Cuts are done on both strands, are very specific and are staggered – The fragment ends are called sticky ends

because they can pair up with a complementary single strand of DNA with the help of DNA ligase

– They are able to form hydrogen bonds with complementary sequences on other DNA molecules

Example of a Restriction EnzymesEcoR1 cuts at 2 different points (at arrows)

EcoR1 cuts DNA into smaller fragments that have sticky ends

Sticky ends hydrogen bond to complementary DNA with DNA ligase

• Fragments cut by same restriction enzyme are usually inserted into a plasmid – which is a small circle of bacterial DNA thus creating recombinant DNA

• The recombinant DNA would then be introduced into a bacteria hoping that the bacterial cells would take up the recombinant DNA

How do we know if the bacteria contained the recombinant DNA?

By many genetic engineering applications

1. Southern Blot

• Technique that uses a probe (specific ordered nucleotide bases complementary to the recombinant DNA)– Extract the DNA– Cut up the DNA– Separate the fragments– Transfer the fragments to a filter sheet– Remove filter and attach probes– Create autoradiograph

autoradiograph

2. Gel Electrophoresis

• Technique which would separate DNA fragments across an electric field

• We are doing this is in AP Lab 9

Gel Electrophoresis – how does it work?

• DNA is negative charged due to the phosphate groups

• So DNA will be put on an electric field and will move toward the positive end

• Small DNA molecules can move through the gel faster than larger ones

• Gives us information about DNA (size of fragment, number of fragment)

3. RFLP’s• Restriction fragment

length polymorphism• Technique used to

determine how closely related the length of DNA fragments are

• Used in forensics to identify a person

• Used to settle disputed parentage

4. PCR

• Polymerase chain reaction

• Technique of amplifying a tiny sample of DNA in a few hours

• Used to duplicate crime scene DNA etc.

13.2

• One goal of recombinant DNA technology is to clone– Produce many identical copies of a particular

DNA sequence– Like PCR does!

Transformation

• Inserting recombinant DNA into a host cell

• The cell or organism that is the host containing the inserted recombinant DNA is called transgenic.

Insulin

• Essential for glucose uptake into cells• Is a good illustration of a medical application to biotechnology• People with type I diabetes cannot make this hormone and must

receive insulin injections• In the past it was made by cattle and pigs, now from E. Coli!• It is a protein of 51 amino acids and is made up of 2 polypeptide

chains• Cattle DNA differs from human DNA by 3 amino acids and pigs just

by 1 amino acid (in the amino acid sequence of insulin)• This difference causes immune responses in diabetics, so these

patients need insulin from humans– Harvesting the protein from deceased humans does not make enough

• So Recombinant DNA biotechnology solves the problem!

Pharming

• Another way of making medically useful products in large amounts

• The production of pharmaceuticals in farm animals or plants

Example

• Human Growth Protein, made in pituitary gland

• People with deficiencies in this are short etc.

• In the past they were treated with protein isolated from the pituitary glands of dead people

• Supply was too limited and a demand for it resulted

• So Recombinant DNA technology was used – bacteria can make the protein, but it is expensive ($30,000 a year)

• So now a transgenic cow is used that secretes the human growth hormone is the milk.

• Only 15 cows are needed the meet the world wide demand of dwarfism

DNA Manipulation Examples

• Larger seeds in plants

• Low fat milk

• Plants that are resistant to disease

• Plants that make their own insecticides

• Corn with high amounts of B-carotene

• Crops that adapt to the environment

• And more!