1. Chapter 6: BIOTECHNOLOGY 4.4 Biotechnological Tools and Techniques

2.  A commitment by the scientific community to determine the location and structure of all genes in the human chromosomes  Scientists sequence the genes of a section of a chromosome and pool the information together.

3.  Having a map of the sequence of nucleotides of human DNA can lead to mapping of genes (listing and finding the locus of each human gene)

4.  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

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

6.  The deliberate manipulation of genetic material  It is possible to move genetic material from one speicies to another because the genetic code is universal ◦ All organisms use the same nitrogenous bases ◦ In all organisms, the same codon codes for the same amino acid (ie: in all species, AUG codes for the amino acid methionine)

7.  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

8.  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

10. STICKY ENDS

11.  STICKY ENDS: both fragments of the newly cleaved DNA have DNA nucleotides lacking complimentary bases.  BLUNT ENDS: The ends of the DNA fragments are fully paired.

12.  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

13.  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

14.  A vector such as a plasmid is obtained  Bacteria carry their DNA in one large circular DNA strand  However, they posses extra DNA in the form of plasmids – circular bits of genetic material carrying ~2-30 genes

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

17.  The same restriction endonuclease must be used on both DNA otherwise the 2 DNAs won’t be able to bind together – there needs to be complimentary base pairing!  DNA Ligase is used to join the cut fragments of DNA together  T4 DNA Ligase – is an enzyme from T4 bacteriophage joins blunt ends together.

18.  http://highered.mcgraw- hill.com/sites/0072437316/student_view0/c hapter16/animations.html# http://highered.mcgraw- hill.com/sites/0072437316/student_view0/c hapter16/animations.html#

19. 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.

20.  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.

21.  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.

22.  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.