DNA Technology

Plasmid Use

• Plasmids are good tools for DNA Technology

• Can be isolated from bacterial cells

• Isolated genes of interest can be inserted into the plasmid.

• How is this insertion done?

• Restriction endonucleases (enzymes)

Restriction Enzymes• Where were restriction enzymes first

found?• Bacterial cells.• They were used to protect bacteria from

intruding phage DNA• Bacterial DNA is modified (methylated

at the restriction sites) to protect it from its own restriction enzymes

• Restriction enzymes often cut DNA leaving “sticky ends”

• Restriction sites are the regions on the DNA that the restriction enzyme cuts.

• Why are restriction sites called palindromes?

How are restriction enzymes

used in DNA technology?

Cloning Genes of Interest

• How can a biologist make large amounts of a gene and thereby produce lots of protein products?

• Clone the genes in recombinant plasmids.

Which method of bacterial genomic

alteration is exploited in

this process?

cDNA

• What is the problem with inserting a human gene into a bacterial plasmid?

• Introns are not spliced in prokaryotes.

• How can this problem be solved?

• Reverse Transcription of mRNA.

Why is cDNA

shorter than the original eukaryotic

DNA?

• Transgenic tobacco plant with gene from jellyfish encoding for glowing protein

• The transgenic pigs, commonly used to study human diseases, would help researchers monitor and trace changes of the tissues during the physical development

• Image:Results of insect infestation on Bt (right) and non-Bt (left) cotton bolls. Source: USDA

• Insert an additional copy of the gene that produces casein to lift the protein content of the milk

• Disrupt the b-lactoglobulin gene to change the composition of the milk. The b-lactoglobulin gene is responsible for producing the protein that causes lactose allergies from cow's milk.

• Insert a human gene that will produce myelin basic protein in the cow's milk. The protein is extracted from the milk and then purified. The myelin basic protein is a possible treatment for multiple sclerosis.

• In a greenhouse of the ARS Vegetable Laboratory in Beltsville, Maryland, plant physiologist Autar Mattoo examines tomato plants genetically engineered to enhance phytonutrient content and longevity of the fruit.

Gel electrophoresis

• How does gel electrophoresis work?• Uses electric charge to separate

molecules based on their size• What charge does DNA have?• Negative• Which sized fragments will move

furthest through the gel?• Smallest ones.

Restriction Fragment Analysis• Genetic markers are regions of DNA

that vary from person to person• Usually located on non-coding regions

of the DNA.• Using restriction enzymes and gel

electrophoresis, DNA of different individuals can be analyzed and compared.

• Extract DNA and treat it with restriction enzymes

How could you detect the differences between these 2 alleles?

Using RFLP Analysis to Detect Harmful Alleles

• Harmful, disease causing alleles usually have identical RFLP’s within a family.

• Once the known RFLP’s for the normal and disease causing alleles are known family members can be tested using electrophoresis and Southern Blot analysis.

VNTR

• A VNTR is variable numbered tandem repeat.

• Tandem repeats are interspersed throughout the genome.

One VNTR is inherited from Each Parent

• Southern blot analysis usually shows 2 different bands one inherited from each parent.

• How could an individual have one band for the VNTR?

• He/She inherited the same sized VNTR from each parent.

Frequency of VNTR’s

• Frequency of allele pattern at a single VNTR has been established for specific sites within the genome.

• What is the probability of matching a 5 locus DNA profile, where each locus is:0.01, 0.02, 0.03, 0.06 and 0.10?

• One in 27.8 million people will randomly match this profile.

• OJ’s profile was of 24 different loci and he matched all 24!

• The odds were 1 in 10 billion.

Determining PaternityWhich child cannot belong to this set of

parents?

Which lane represents the father?

5C

hild

Mom

Rape Investigation

Did the suspect commit the crime?