Today: Biotechnology

Over 600 recent transposon insertions were identified by examining DNA from 36 genetically diverse humans.

Tbl 1 Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in Genetics 23: 183-191

DNA fingerprinting using RFLPs

Visualizing differences in DNA sequence by using restriction enzymes

Sequence 1

Sequence 2

Restriction Enzymes cut DNA at specific sequences

Fig 18.1

EnzymeRecognitionSequence Cut

EcoRI 5'GAATT C3'CTTAAG

5'---G AATT C---3'3'---CTTAA G--- 5'

BamHI 5'GGATCC3'CCTAGG

5'---G GATCC---3'3'---CCTAG G---5'

HindIII 5'AAGCTT3'TTCGAA

5'---A AG CTT---3'3'---TTCGA A---5'

TaqI 5'TCGA3'AGCT

5'---T CGA---3'3'---AGC T---5'

AluI 5'AGCT3'TCGA

5'---AG CT---3'3'---TC GA---5'

Examples of some restriction enzymes…tbl 18.3

Visualizing differences in DNA sequence by using restriction enzymes

Sequence 1

Sequence 2

Fig 20.5+.6

Separating DNA on a gel by sizeFig 20.6

• Gel electrophoresis Fig 24.21

The different sized bands can arise from different cut sites and/or different number of nucleotides between the cut sites.

Fig 22.23Sequence 1

Sequence 1

Sequence 2

Sequence 2

DNA fingerprinting

DNA fingerprinting

DNA fingerprinting

Can DNA be obtained from hair?

How can DNA be obtained from such a small sample?

The inventor of PCR

Polymerase Chain Reaction:amplifying DNA

Fig 18.6

Polymerase Chain Reaction

Fig 18.6

Polymerase Chain Reaction:Primers allow specific regions to be amplified.

Fig 18.6

The inventor of PCR

PCR animation http://www.dnalc.org/ddnalc/resources/pcr.html

Areas of DNA from very small samples can be amplified by PCR, and then cut with

restriction enzymes for RFLP analysis.

Genetic Engineering: Direct manipulation of DNA

Fig 18.2

Bacteria can be modified or serve as intermediates

Fig 18.2

a typical bacteria

Bacterial DNA

plasmid DNA

A typical bacterial plasmid used for genetic engineering

tbl 18.2

Moving a gene into bacteria via a plasmidFig 18.2

Bacterial DNA

plasmid DNA

What problems exist for expressing eukaryotic gene in bacteria?

Reverse transcriptase can be used to obtain coding regions without introns.

Fig 18.4

After RT, PCR will amplify the gene or DNA

Fig 18.6

Moving a gene into bacteria via a plasmid

RT and PCR

Fig 18.2

Restriction Enzymes cut DNA at specific sequences

Fig 18.1

Restriction enzymes cut DNA at a specific sequence

Fig 18.1

Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends

Fig 18.1

A typical bacterial plasmid used for genetic engineering

Using sticky ends to add DNA to a bacterial plasmidFig 18.1

Transformation of bacteria can happen via several different methods.

tbl 6.1

Bacteria can take up DNA from the environment

Fig 9.2

Tbl 6.1

Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane:

•Electroporation

•Heat shock

•Osmotic Stress

How can you know which bacteria have been transformed, and whether they have the insert?

Fig 18.1

Resistance genes allow bacteria with the plasmid to be selected.

Bacteria with the resistance gene will survive when grown in the presence of antibiotic

Fig 20.5 Is the insert present?Plasmids with the MCS in the lacZ gene can be used for blue/white screening…

Fig 18.1

A typical bacterial plasmid used for genetic engineering

Intact lacZ makes a blue color when expressed and provided X-galactose

When the lacZ gene is disrupted, the bacteria appear white

Blue/white screening:

Transformed bacteria plated on antibiotic and X-gal plates.Each colony represents millions of clones of one transformed cell.

Fig 18.1

Successful transformation will grow a colony of genetically modified bacteria

Fig 18.1

Inserting a gene into a bacterial plasmid

RT and/or PCR

Fig 18.1

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Texas =70 ha

Bacteria can be used to transform plantsGlobal area planted with GM crops

http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html