Today: Biotechnology Exam #2 Th 10/23 in class

Map of human chromosome 20 Your DNA Map of human chromosome 20 http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?ORG=human&CHR=X&MAPS=ideogr[Xpter:Xqter],genes[1.00:153692391.00]

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

Restriction Enzymes cut DNA at specific sequences Fig 18.1 Restriction Enzymes cut DNA at specific sequences

Examples of some restriction enzymes… tbl 18.3 Examples of some restriction enzymes…

Visualizing differences in DNA sequence by using restriction enzymes Fig 20.5+.6 Visualizing differences in DNA sequence by using restriction enzymes Sequence 1 Sequence 2

Separating DNA on a gel by size Fig 20.6 Separating DNA on a gel by size

Fig 24.21 Gel electrophoresis

The different sized bands can arise from different cut sites and/or different number of nucleotides between the cut sites. Sequence 1 Sequence 2 Sequence 1 Fig 22.23 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: amplifying DNA

Polymerase Chain Reaction Fig 18.6 Polymerase Chain Reaction

Polymerase Chain Reaction: Fig 18.6 Polymerase Chain Reaction: Primers allow specific regions to be amplified.

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 A typical bacterial plasmid used for genetic engineering

Moving a gene into bacteria via a plasmid Fig 18.2 Moving a gene into bacteria via a plasmid

What problems exist for expressing eukaryotic gene in bacteria? Bacterial DNA plasmid DNA

Fig 18.4 Reverse transcriptase can be used to obtain coding regions without introns.

After RT, PCR will amplify the gene or DNA Fig 18.6 After RT, PCR will amplify the gene or DNA

Moving a gene into bacteria via a plasmid Fig 18.2 Moving a gene into bacteria via a plasmid RT and PCR

Restriction Enzymes cut DNA at specific sequences Fig 18.1 Restriction Enzymes cut DNA at specific sequences

Restriction enzymes cut DNA at a specific sequence 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

A typical bacterial plasmid used for genetic engineering

Using sticky ends to add DNA to a bacterial plasmid Fig 18.1

Fig 18.1 If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself.

Fig 18.1 The plasmid is treated with phosphatase to remove the 5’-P, preventing self-ligation

Transformation of bacteria can happen via several different methods. tbl 6.1 Transformation of bacteria can happen via several different methods.

Bacteria can take up DNA from the environment Fig 9.2

Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Electroporation Heat shock Osmotic Stress Tbl 6.1

Fig 18.1 How can you know which bacteria have been transformed, and whether they have the insert?

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

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: Fig 18.1 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

Inserting a gene into a bacterial plasmid RT and/or PCR Fig 18.1 Inserting a gene into a bacterial plasmid

Bacteria can be used to transform plants Global area planted with GM crops Texas = 70 ha Millions of Hectares http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15b

Agrobacterium infect plants, inserting their plasmid DNA into the plants genome. Fig 19.15

By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant. Fig 19.16

The generation of a transgenic plant Fig 19.16 The generation of a transgenic plant Grown on herbicide