Gene Technology 1

Biotechnology

You are only responsible for the material we get through in class

End of Chapter questions: Understand: 1,2,4,5,7, Apply: all Synthesize: 1

Do all mQuiz questions

Chapter 17

Gene Technology 2

What are the methods of genetic engineering ?

DNA cleavage

Restriction endonucleases

“sticky ends”

Gene Technology 3

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Gel electrophoresis

Used to analyze DNA fragments

Agarose – acts like a sieve

DNA has negative charge

Gene Technology 4

How can one determine which bands have a particular sequence?

Southern blotting -- involves 3 key steps

1. Transfer to nitrocellulose

2. “Probe” with 32P labeledDNA sequence

(e.g., “AATGGCAGGT”)

3. Perform autoradiography

Gene Technology 5

DNA fragments are manipulated (e.g., ‘cloned’) by inserting into a ‘vector’

A ‘vector’ is a carrier -- Plasmids or viruses

“Cloning” means to copy

Plasmids with special genes are used

Gene Technology 6

How can a “genomic library” be created? -- Cells with DNA fragments from entire genome

1. Cut up DNA into pieces

2. insert pieces at random into vector

3. “Transfect” into bacteria

Problems, however: -- Few bacteria receive a

plasmid-- Not all plasmids contain a

DNA fragment

Also…Very few plasmids contain DNA

with any particular gene

Cloning Steps

Gene Technology 7

How to screen for recombinant plasmids?

Cells grown on Medium containing ampicillin and Xgal

Form blue or whitecolonies

Gene Technology 8

How to screen for cells containing a specific DNA fragment?

Plate library

Make replica onNitrocellulose

Probe with Radio-labeled“DNA probe”

Perform autoradiography

Find original colony

Gene Technology 9

How can the DNA for a particular gene be isolated from original organism?

-- one way is a “cDNA” (complementary DNA)

Starts with mRNA

Reverse transcriptase

DNA polymerase

cDNA lacks intron sequences

Gene Technology 10

What can cloned genesbe used for?

Transferring to other organisms, e.g. -- insulin production to bacteria-- herbicide/ insect resistance to plants-- crop yield & nutritional value (e.g. ‘golden rice’)-- biofuel (e.g., rape seed oil)

Gene therapy … some day-- cystic fibrosis-- hemophilia-- various cancers-- many others

Gene sequencing -- evolutionary relationships-- protein structure predications

Sheep engineered to produce the human protein alpha-1-antitrypsin (AAT)

in milk for treatment of emphysema

Control vs Roundup resistant alfalfa in field treated with Roundup

Gene Technology 11

“DNA Fingerprinting”

Used to identify source of DNA

Small genetic differences createRestriction Fragment Length Polymorphisms(RFLPs)

Forensic samples (‘CSI Biology’)

-- blood, semen, saliva

How are RFLPs used to createa DNA fingerprint? DNA fingerprint

Gene Technology 12

35 cycles = 235 = 68 billion copies PCR

PCR is used to analyze very small amounts of DNA

PCR = Polymerase Chain ReactionRequires:ThermocyclerPrimersTaq-polymerase

PCR animation