1. 4/26/2010
BIOTECHNOLOGY

2. Recombinant DNA
Recombinant DNA: a fragment of DNA composed of sequences originating from at least 2 different sources
Why? – ex: Agricultural Benefits: increase in crop yields; disease resistance; crop longevity

3. Genetic Engineering HOW?? Hypothetically…..
If we could insert a gene into another organism’s genome (DNA), that organism would express that gene (make the protein the gene codes for)
To do this, we would need “molecular scissors” to cut the gene sequence from our original source and “molecular glue” to insert the gene sequence into the host organism’s DNA

4. Restriction Endonucleases
Also known as Restriction Enzymes are “molecular scissors” that can cleave (cut) double stranded DNA at a specific base-pair sequences.
Bacterial enzymes
Recognition Site: the specific sequence where the restriction enzyme makes its cut.
Usually palindromic
~4 to 8 nucleotides long

6. STICKY ENDS

7. BLUNT ENDS: The ends of the DNA fragments are fully paired.
STICKY ENDS: both fragments of the newly cleaved DNA have DNA nucleotides lacking complimentary bases.
Sticky end fragments make use of hydrogen bonding, thus facilitating the initial joining of fragments that then are completely joined by another enzyme
BLUNT ENDS: The ends of the DNA fragments are fully paired.
Blunt ends have a significantly lower yield and have the potential to insert DNA in the opposite orientation desired

8. Can easily be used to create recombinant DNA
Restriction enzymes that produce sticky ends are more useful because these DNA fragments can easily be joined to other DNA sticky ends fragments made by the same restriction enzyme.
Can easily be used to create recombinant DNA

9. Plasmids for Genetic Engineering
A desired sequence of nucleotides from source DNA can be cut out of the genome, separated and annealed to another DNA molecule These DNA fragments can be inserted into bacterial plasmids Plasmids are small, circular, double stranded DNA molecules found in bacterial cells These plasmids are independent of the chromosome of the bacteria The DNA contained in plasmids can be replicated and expressed When foreign DNA is taken up by a bacterial cell via plasmid or virus this is known as TRANSFORMATION.

10. Restriction sites
The gene we want to introduce
The DNA that we want to insert the gene into
The gene is cut from its original DNA strand

11. Application – The Ti Plasmid

12. DNA Ligase
The same restriction endonuclease (enzyme) must be used on both DNA otherwise the 2 DNAs won’t be able to bind together – there needs to be complimentary base pairing (the sticky ends must match up)
Two complimentary strands will naturally anneal and form hydrogen bonds between the base pairs but an enzyme is required to form the phosphodiester backbone of the DNA

13. DNA Ligase is used to join the cut fragments of DNA together via a condensation reaction. Phosphodiester bonds are reformed between adjacent bases
T4 DNA Ligase – is specific enzyme that is especially useful for joining blunt ends together

14. Technique for Gene Transfer
Materials Required:
A vector (used to carry the gene into the host cell)
A host cell (which will express the gene – make the protein)
Restriction enzymes
DNA ligase –(used to join together DNA blunt or sticky ends)

15. Animation Frog experiment

16. Where DO RESTRICTION ENZYMES COME FROM?

17. Restriction enzymes act as an immune system in the bacterium
Restriction enzymes act as an immune system in the bacterium. When a bacteriophage (a virus) tries to inject its DNA into the bacteria, restriction enzymes cut up the bacteriophage DNA into many fragments – thus, preventing it from doing any harm to the bacterium.

18. Methylases
Restriction endonucleases must be able to distinguish between foreign DNA and their own DNA otherwise they would cut up their own DNA.
METHYLASES are enzymes that modify a restriction site by adding a methyl group to and preventing the restriction endonuclease from cutting it. This prevents the cell from cutting its own DNA methylation
Foreign DNA in a cell will not be methylated and therefore may be broken down by the enzymes

19. Real Life Application: INSULIN
Insulin is a hormone that regulates blood sugar levels by converting excess glucose into glycogen for long term storage.
Diabetics do not make sufficient amounts of insulin.
Diabetics may be required to take insulin injections.
Using restriction enzymes, the gene for synthesizing insulin is cleaved out of DNA and inserted into the DNA plasmid of a nonharmful bacteria.
DNA ligase is added to the bacteria.
The bacteria is also given the necessary amino acids.
The recombinant DNA will express the insulin gene and make insulin that can be collected and administer to diabetics

20. Human Genome Project
Scientists worked to sequence a chromosome, one gene at a time and pooled the information together and store this information in database.
The aim was to;
determine the sequences of the 3 billion chemical base pairs that make up human DNA.
identify all the approximate 30,000 genes in human DNA.
Have a map of the sequence of nucleotides of human DNA that could lead to mapping of genes (listing and finding the locus of each human gene)

21. Human Genome Project: OUTCOME
Improved understanding of genetic diseases
Production of medicines (based on DNA sequences) to cure diseases
Genetic screening (and preventative medicine)
Focused research
Provides more information about the evolutionary paths between species

22. Human Genome Project
NOVA Online | Cracking the Code of Life | Watch the Program Here
genome.gov | Education Kit - Component DOWNLOAD Page
Sequence for yourself