2.  It is important to know the order of nucleotides in a strand of DNA  The process of DNA sequencing (determining the order of nucleotides) uses processes found in vivo (inside the cell)  Recall that DNA polymerase adds complementary nucleotides to a single strand of DNA  The nucleotides are found in high concentration in the cell

4.  Scientists attempt to mimic this process in vitro (in a lab)  A length of DNA is first heated ◦ This separates the two strands  Then, a heat-resistant DNA pol is added  Ordinary A, C, T and G nucleotides are added in high concentration  However, a special type of each nucleotide is also added in low concentration  Two features make them unique 1.They are fluorescently labeled 2.They lack two hydroxyl groups on carbon 2 and 3 (called dideoxy)

6.  The label allows them to be detected when ran through a gel (more on this later)  The missing hydroxyls stops DNA pol from adding any more nucleotides past that point  The addition of the modified nucleotides is always random  Eventually, a set of labeled strands of all lengths is generated ◦ Each ends with a dd nucleotide (one of four possible colours)  These are ran through a gel, arranging them in order of size

8.  The gel is passed through a detector, determining the order of the nucelotides

9.  Sometimes, scientists want to make copies of a strand of DNA ◦ For example, a small amount of DNA left at a crime scene  This can be done in vitro through a polymerase chain reaction  Involves a three step cycle 1.Heat to separate strands 2.Cooling to allow annealing of DNA primers 3.Addition of nucleotides via DNA pol

12.  http://www.youtube.com/watch?v=HMC7c2T 8fVk http://www.youtube.com/watch?v=HMC7c2T 8fVk  http://www.youtube.com/watch?v=_YgXcJ4n -kQ http://www.youtube.com/watch?v=_YgXcJ4n -kQ

13.  Recall that restriction enzymes make “sticky ends”  This works with any organism that has DNA as its genetic material  As a result, sections of DNA from one organism can be spliced into another  Bacteria is often used since it has plasmids ◦ These are small, circular strands of DNA ◦ Easy to remove  The original version is called a cloning vector

14.  Plasmids are easily removed and reinserted  We have used these cloning vectors to engineer bacteria to produce proteins such as insulin and growth hormone

15. Recombinant DNA Gene for human growth hormone Human Cell Bacteria cell Bacterial chromosome Plasmid Sticky ends DNA recombination Bacteria cell containing gene for human growth hormone DNA insertion

16.  DNA that has been modified by adding foreign segments is called recombinant

17.  It is also possible to transform multicellular organisms such as plants and animals  To do so, microorganisms, such as bacteria and viruses, must be used  For example, a bacteria that causes tumors in plants can be altered so the tumor gene is replaced by a useful gene  However, these methods can only work if the microorganism can insert its DNA into a host

18. Complete plant generated from transformed cell. Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome. Plant cell colonies Transformed bacteria introduce plasmids into plant cells. Agrobacterium tumefaciens Cellular DNA Gene to be transferred Recombinant plasmid