1. Recombinant Technololgy
Exploring Genes
Recombinant Technololgy

2. Restriction Enzymes
What are restriction enzymes and how are they used?
enzymes that recognize specific base sequences in DNA and cleave the DNA at those sequences
cuts DNA into specific fragments for analysis

3. Restriction Enzymes
What characteristic do the cleavage sites of restriction enzymes share?
palindromic

4. Restriction Enzymes
How do we identify a particular restriction enzyme?
based upon host organism from which enzyme was isolated
first letter genus
second two letters species

5. Restriction Enzymes
What is a restriction digest and how is it analyzed?
DNA cut by one or more restriction enzymes
gel electrophoresis

6. Restriction Enzymes What is Southern blotting and how is it used?
separating mixture of restriction fragments of DNA by electrophoresis and probing with labeled DNA

7. Restriction Enzymes What are Northern blots? What are Western blots?
separation of RNA fragments and probing with labeled DNA
What are Western blots?
separation of proteins and probing with labeled antibody

8. DNA Sequencing
How is controlled termination of DNA replication used to determine the sequence of a DNA molecule?

9. DNA Sequencing

10. DNA Sequencing How large are complete genomes?
X174 virus – 5386 base pairs (bp)
human mitocondrial DNA – 16, 569 bp
Haemophilus influenzae – 1,830,137 bp
C. elegans – 100,000,000 bp
H. sapiens – 3,000,000,000 bp

11. DNA Synthesis How can DNA be synthesized in the lab?
solid-phase synthesis by phosphite triester method

12. DNA Synthesis
What does the activated monomer look like?

13. DNA Synthesis Why is the ability to synthesize DNA chains valuable?
making radioactive or fluorescent probes helps to locate presence of genes
synthetic probe can be used as primer
can synthesize customized genes

14. Polymerase Chain Reaction
What is PCR?
technique used to make many copies of a specific DNA sequence
Why is this a valuable tool?
amplify a small amount of DNA
What materials are needed?
primers
all dNTP’s
heat-stable DNA polymerase

15. Polymerase Chain Reaction

16. Polymerase Chain Reaction
What are some of the ways PCR has been used
forensics
paternity suits
detection of HIV
cancer detection
detection of tuberculosis bacillus

17. Recombinant DNA What is recombinant DNA?
novel DNA molecules
What is needed to make recombinant DNA?
vector
plasmid
virus

18. Recombinant DNA
What else?
restriction enzyme
DNA ligase

19. Recombinant DNA

20. Recombinant DNA
Any DNA molecule can be made to contain sticky or cohesive ends

21. Cloning Vectors
Plasmids and bacteriophages serve as useful cloning vectors
example – pBR322

22. Cloning Vectors
Lambda phage can destroy host cell or become incorporated into host cell.

23. Cloning Vectors How are phages used as vectors? What advantages
do these modified viruses have over plasmids?

24. Cloning Vectors
What are some of the advantages of using M13 phage as a cloning vector?
exists as a single-stranded molecule
doesn’t kill bacterial host
can grow large quantities of M13
can sequence foreign DNA cloned into M13 with dideoxy method

25. Cloning Vectors
How is M13 used for cloning?

26. Recombinant DNA What is a genomic library and how is it prepared?
collection of genomic DNA fragments inserted into a cloning vector

27. Recombinant DNA
How does one screen a genomic library to find which phages contain a particular gene?
grow phage on lawn of bacteria
plaques form where phage infect cells
prepare replica on nitrocellulose sheet
treat with NaOH to lyse bacteria and denature DNA
hybridize with 32P labeled probe
audoradiography

28. Recombinant DNA What are cosmids?
combinations of plasmids and lambda phages
can hold 45 kb inserts
What are bacterial artificial and yeast artificial chromosomes (BAC &YAC)?
pieces of DNA containing centromere, autonomous replication sequence (ARS), and telomeres
can hold kb inserts

29. Recombinant DNA What is chromosome walking and how is it used?
technique of sub-cloning and re-screening used to analyze long stretches of DNA

30. Recombinant DNA
What is c-DNA?
DNA made from m-RNA
How is it made?

31. Recombinant DNA How is c-DNA used?
placed in vectors, inserted into bacteria, forms a c-DNA library
How does a c-DNA library differ from a genomic DNA library?
c-DNA contains no introns

32. Recombinant DNA
How are c-DNA clones screened?

33. Recombinant DNA For what purpose are gene chips used?
to measure the level of gene expression in eukaryotic cells via fluorescence

34. Recombinant DNA
Why must many eukaryotic genes be expressed in eukaryotic cells?
require posttranslational modification
What is the most effective way of introducing eukaryotic DNA into host cells?
retroviruses – Moloney murine leukemia virus
Baculovirus – insect cells
Vaccinia virus

35. Recombinant DNA What are transgenic mice?
mice containing and expressing foreign DNA

36. Recombinant DNA
What led to the formation of this oversized mouse?

37. Recombinant DNA
What is homologous recombination and how has it been used to cause gene knockouts?

38. Recombinant DNA How can foreign genes be inserted into plant cells?
Ti plasmids
effective in dicots and a few monocots

39. Recombinant DNA How can foreign genes be inserted into plant cells?
electroporation
cereal monocots and dicots
“gene guns”
DNA coated onto tungsten pellets
fired at cells

40. Recombinant DNA
How can one construct a new gene by producing deletions?
cut plasmid at two sites with restriction enzyme and ligate to form smaller plasmid
cut plasmid at one site, use endonuclease to remove additional bases, and ligate

41. Recombinant DNA What is oligonucleotide-directed mutagenesis?
used to replace code for particular amino acid
prepare oligonucleotide primer containing a mutant code for amino acid to be changed
separate plasmid strands, anneal primer, allow for replication
some progeny will contain mutation

42. Recombinant DNA
What is cassette mutagenesis?
type of insertion

43. Recombinant DNA What are some of the benefits of this technology?
understanding protein structure, mechanisms of catalysis, etc.
produce large amounts of useful proteins
insulin
growth hormone
drugs
diagnostic reagents
gene therapy
agriculture