Gene Technology

At the beginning:• Studies using interferon:

– Interferon increases human resistance to viral infection and scientists were interested in its possible usefulness in cancer therapy.

– It was rare in the blood and needed in large quantities for study.

– Interferon gene introduced into bacteria genome.

– Bacteria divide rapidly so began to produce interferon in large quantities.

At the beginning:• Studies using interferon:

– Interferon increases human resistance to viral infection and scientists were interested in its possible usefulness in cancer therapy.

– It was rare in the blood and needed in large quantities for study.

– Interferon gene introduced into bacteria genome.

– Bacteria divide rapidly so began to produce interferon in large quantities.

A bacteria

Bacterial Reproduction

Genetic Engineering

• The bacteria produced a line of genetically altered cells from a single altered cell = cloning.

• Insulin gene can also be “grown” this way.

• This marked the beginning of genetic engineering.– The ability to cut DNA into

recognizable pieces and rearrange them in different ways.

How To:

• Cut the source of the DNA that carries what you want and insert into a plasmid or infective virus.

A Virus

Getting In

Taking Over

In order to make it work:• MUST• 1. cut the source DNA and the plasmid DNA so that the desired fragment

can be spliced.– Done by enzymes the recognize and cleave (cut) specific sequences of

nucleotides.

Restriction Enzyme

Source Recognition Site

BAMHI Bacillus amyloliquefaciens G GATC CC CTAG G

EcoRI Eschirichia Coli R factor G AATT CC TTAA G

HaeIII Hemophilus aegyptus GG CCCC GG

HindIII Hemophilus influenzae A AGCT TT TCGA A

TaqI Thermus aquaticus T CG AA GC T

Restriction Endonucleases• Bacteria must defend themselves against bacteriophages (viruses that specifically

attack bacteria).• Through the lytic cycle the viruses can attack, kill, and take over many more

bacteria.• To defend themselves bacteria contain enzymes called restriction endonucleases.• These recognize specific nucleotide sequences in a DNA strand, bind to them,

and cleave them at the recognition sequence.• To keep these enzymes from attacking their own DNA bacteria add methyl (using

mehylases) to the recognition sites so the endonucleases can’t bind.

Restriction Endonucleases• Bacteria must defend themselves against bacteriophages (viruses that

specifically attack bacteria).• Through the lytic cycle the viruses can attack, kill, and take over many more

bacteria.• To defend themselves bacteria contain enzymes called restriction

endonucleases.• These recognize specific nucleotide sequences in a DNA strand, bind to them,

and cleave them at the recognition sequence.• To keep these enzymes from attacking their own DNA bacteria add methyl

(using mehylases) to the recognition sites so the endonucleases can’t bind.

Restriction endonuclease

How To Cut the DNA

• Find the palindromes.– Nucleotides at one end of the recognition sequence

are comlementary to those at the other end so they have the same nucleotide sequence running in opposite directions.• Allows it to cut across two strands• Since the cleave site isn’t directly centered each fragment

has a single stranded end a few nucleotides long.• These fragments are complementary to each other and

can recombine using DNA ligase (the spotwelder).– These are considered “sticky” ends and will attract their

complementary base pair sets.

Cutting Activity

Restrictions Endonucleases Cut

• Hundreds of restiction enzymes – Each with own recognition sequence

• By chance the recognition site will arise within a given sample. – The shorter the sequence the more often it will probably occur.– Once two fragments attach, they can be joined by DNA ligase

to reform the phosphodiester bonds of DNA.– ANY two fragments produced by the same restriction

endonuclease can be joined together. Elephant DNA cleaved by an endonuclease can be joined to ostrich DNA if cleaved by the same endonuclease.

EcoRI

• Cleaves sequence GAATTC between the G and the A.

• Since they run in opposite directions, they are complementary… sticky.