1. Recombinant DNA Technology

2. Review What are enzymes? What function do they serve?
What makes up the DNA sequence?
How is the genetic code determined in an organism?

3. Recombinant DNA Technology

4. Discovery of restriction enzymes
Bacteriophages infect bacteria by inserting their genetic material (DNA) into the DNA of bacteria
In the 1960s, microbiologists discovered that some bacteria are protected from bacteriophage infection because they can restrict phage replication
Scientists proposed that restricted growth of phages occurred cause the bacteria contained enzymes that could cut viral DNA into small pieces and prevent replication
These enzymes were called restriction enzymes

5. Restriction Enzymes Primarily found in bacteria
Given names based on genus and species names of bacteria from which they are isolated
Cut DNA by cleaving the phosphodiester bond (in sugar-phosphate backbone) that joins nucleotides in DNA strand
Show specificity for certain DNA substrates
Enzymes bind to, cut (digest) DNA within specific sequences of bases called recognition sequence or restriction site
Typically recognize 4, 6, or 8 nucleotide sequences

6. Restriction Enzymes
Restriction enzyme EcoRI, named because it was discovered in Escherichia coli

7. Restriction Enzymes
Some restriction enzymes cut DNA to create DNA fragments with overhanging single-stranded ends called “sticky” or cohesive ends

8. Table 3.1 – Cohesive Ends

9. Some enzymes generate fragments with non-overhanging or blunt ends
Restriction Enzymes
Some enzymes generate fragments with non-overhanging or blunt ends

10. Restriction Enzymes
Enzymes that produce cohesive ends are often favored over blunt-end cutters because DNA fragments with cohesive ends can easily be joined together.
DNA from any source, bacteria, humans, dogs, frogs, dinosaurs, and ancient human remains can be digested by a particular restriction enzyme as long as it has the restriction site for that enzyme!

12. Restriction Enzymes DNA cutting enzymes Very specific
Cuts double stranded DNA
Two incisions to cut DNA at sugar-phosphate bond on each strand
Very specific
Enzymes recognize short nucleotide sequences
Enzymes cut the DNA at specific points within DNA
For example – if the restriction enzyme cuts between a C and G then
5'GGCC3' 3'CCGG5'
5'GG CC3' 3'CC GG5'
5'GG CC3' 3'CC GG5'

13. Restriction Enzymes
Each restriction enzyme recognizes a specific restriction site, a short nucleotide sequence
Some restriction enzymes cut straight across the DNA
Some restriction enzymes cut at offset nucleotides
For example -
G A A T T C
C T T A A G
G A A T T C
C T T A A G
G AATC
CTTAA G

14. Restriction Enzymes
Sequences that are cut at offset points produce overhanging pieces of DNA called “sticky ends”
Sticky end
G AATC
CTTAA G

15. Restriction Enzymes

16. Restriction Fragments
Each cut of DNA creates restriction fragments
A long strand of DNA may be cut several times by a restriction enzyme to create several restriction fragments

17. Why are restriction fragments useful?
Recombinant DNA
Gene isolation
Genome mapping
Gel electrophoresis
Genetic engineering
Gene therapy

19. Recombinant DNA Technology
Technique that allows DNA to be combined from different sources

20. Recombinant DNA/Restriction Enzyme Example
In the early 1970s, a group of scientists joined together DNA from E. coli chromosomes and DNA from a primate virus
They isolated the DNA from each organism and cut them into fragments with EcoRI
They added the E. coli and viral DNA fragments to a reaction tube and added DNA ligase
They created a hybrid of the two DNA types, recombinant DNA

21. Recombinant DNA Technology
Plasmids are considered extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosome
Plasmids can be used as vectors, pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA

22. Recombinant DNA Technology
Plasmids are considered extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosome
Plasmids can be used as vectors, pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA

23. Plasmid Vectors – used to clones specific DNA

24. NIH and RAC Scientists were concerned about:
with what might happen if recombinant bacteria were to leave the lab
if such bacteria could transfer their genes to other cells
survival in other organisms including humans
National Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC) to evaluate the risks of recombinant DNA technology and establishing guidelines

25. Transformation
Transformation – process for inserting foreign DNA into bacteria
Treat bacterial cells with calcium chloride solutions
Add plasmid DNA to cells chilled on ice
Briefly heat the cell and DNA mixture, plasmid DNA will enter the bacterial cell
Once inside bacteria, plasmids replicate and express their genes

27. Selection
Joining of DNA fragments and plasmids by transformation can be inefficient
Some plasmids will rejoin and recircularize and not bond with any foreign DNA
During transformation, many cells will not take up DNA
Recombinant bacteria, those transformed with a recombinant plasmid, must be separated from nontransformed bacteria as well as cells with plasmids without foreign DNA
This process is called selection because it is designed to identify (select for) recombinant bacteria and prevent the growth of nontransformed bacteria and bacteria without foreign DNA

30. Polymerase Chain Reaction
Technique for making copies or amplifying a specific sequence of DNA in a short period of time.

31. PCR (Polymerase Chain Reaction)
1. Target DNA to be amplified is added to a thin-walled tube and mixed with deoxyribonucleotides (dATP, dCTP, dGTP, dTTP), buffer and DNA polymerase. Primers are added to the mixture; they are complimentary to nucleotides on opposite ends of the target DNA.
2. Tube is placed in a thermal cycler (gene cycler). This will take the sample through a series of reactions called the PCR cycle.

32. Thermal Cycler
Denaturation – tube is heated to ~94-96ºC separating DNA into single strands
Hybridization (annealing) – tube is cooled to ~60-65ºC allowing primers to bond to ends of DNA
Extension (elongation) – temperature raised slightly and DNA polymerase copies the DNA by binding to primer and making a new strand
DNA polymerase used in this reaction is Taq DNA polymerase from Thermus aquaticcus. Bacteria is adapted to live in hot springs it has enzymes that can withstand high temperatures necessary for PCR.

34. PCR At the end of one cycle, amount of DNA has doubled.
Researchers can repeat cycle (usually times) to amplify millions of copies of DNA

35. Uses of PCR Studying gene expression
Detection of viral or bacterial infections
Diagnosing genetic conditions
Amplifying trace amounts found at crime scenes
Amplifying trace amounts found in ancient DNA

36. Gel Electrophoresis

37. Gel Electrophoresis
Used to separate and visualize DNA fragments based on size
Agarose gel is a semisolid material with small pores through which DNA can travel; fragments are separated by size – smaller fragments can travel further through pores than larger fragments

38. Gel Electrophoresis
1. To run a gel, it is submerged in buffer solution that will conduct electricity
2. DNA samples are loaded into small wells and an electric current is passed through the gel
3. The sugar-phosphate backbone renders DNA with a negative charge; therefore DNA will move to the positively charged end
Because migration distance is inversely proportional to the size of the DNA fragment, large DNA fragments migrate short distances and small fragments migrate faster
Dyes are added to monitor DNA migration

40. Gel Electrophoresis Animations