1. Recombinant DNA and Genetic Engineering
Chapter 15

2. Genetic Changes
Humans have been changing the genetics of other species for thousands of years
Artificial selection of plants and animals
Natural processes also at work
Mutation, crossing over

3. Genetic Engineering
Genes are isolated, modified, and inserted into an organism
Made possible by recombinant technology
Cut DNA up and recombine pieces
Amplify modified pieces

4. Discovery of Restriction Enzymes
Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack
Discovered bacteria have an enzyme that chops up viral DNA

5. Specificity of Cuts Restriction enzymes cut DNA at a specific sequence
Number of cuts made in DNA will depend on number of times the “target” sequence occurs

6. Making Recombinant DNA5’ G
A A T T C 3’
C T T A A G
one DNA fragment
another DNA fragment 5’ G
A A T T C 3’ 3’
C T T A A G 5’

7. Making Recombinant DNA
nick 5’ G
A A T T C 3’ 3’
C T T A A G 5’
nick
DNA ligase action G
A A T T C
C T T A A G

8. Using Plasmids Plasmid is small circle of bacterial DNA
Foreign DNA can be inserted into plasmid
Forms recombinant plasmids
Plasmid is a cloning vector
Can be used to deliver DNA into another cell

9. Using Plasmids DNA fragments + enzymes recombinant plasmids
host cells containing recombinant plasmids

10. Making cDNA mRNA transcript mRNA–cDNA hybrid single-stranded cDNA
double-stranded cDNA

11. Amplifying DNA
Fragments can be inserted into fast-growing microorganisms
Polymerase chain reaction (PCR)

12. Polymerase Chain Reaction
Sequence to be copied is heated
Primers are added and bind to ends of single strands
DNA polymerase uses free nucleotides to create complementary strands
Doubles number of copies of DNA

13. Polymerase Chain Reaction
DNA to be amplified
DNA is heated
Primers are added

14. a PCR starts with a fragment
of double-stranded DNA
b The DNA is heated to 90°–
94°C to unwind it. The single
strands will be templates
c Primers designed to
base-pair with ends of the
DNA strands will be mixed
with the DNA
d The mixture is cooled. The
lower temperature promotes
base-pairing between the
primers and the ends of the
DNA strands
e DNA polymerases recognize
the primers as START tags. They
assemble complementary
sequences on the strands. This
doubles the number of identical
DNA fragments
Fig. 15.6a, p. 226

15. Polymerase Chain Reaction
Mixture cools
Base pairing occurs
Complementary
strand synthesized

16. Primers Short sequences that DNA polymerase recognizes as start tags
To carry out PCR, must first determine nucleotide sequences just before and after the gene to be copied
Complementary primers are then created

17. The DNA Polymerase
Most DNA polymerase is denatured at high temperature
Polymerase used in PCR is from bacteria that live in hot springs

18. Temperature Cycles DNA is heated to unwind strands
Cooled to allow base-pairing with primers and complementary strand synthesis
DNA is heated again to unwind strands
Cycle is repeated over and over again

19. DNA Fingerprints Unique array of DNA fragments
Inherited from parents in Mendelian fashion
Even full siblings can be distinguished from one another by this technique

20. Tandem Repeats
Short regions of DNA that differ substantially among people
Many sites in genome where tandem repeats occur
Each person carries a unique combination of repeat numbers

21. RFLPs Restriction fragment length polymorphisms
DNA from areas with tandem repeats is cut with restriction enzymes
Because of the variation in the amount of repeated DNA, the restriction fragments vary in size
Variation is detected by gel electrophoresis

22. Gel Electrophoresis DNA is placed at one end of a gel
A current is applied to the gel
DNA molecules are negatively charged and move toward positive end of gel
Smaller molecules move faster than larger ones

23. Analyzing DNA Fingerprints
DNA is stained or made visible by use of a radioactive probe
Pattern of bands is used to:
Identify or rule out criminal suspects
Determine paternity

24. Genome Sequencing
Sequence of bacterium Haemophilus influenzae determined
Automated DNA sequencing now main method
3.2 billion nucleotides in human genome determined in this way

25. Nucleotides for Sequencing
Standard nucleotides (A,T,C, G)
Modified versions of these nucleotides
Labeled so they fluoresce
Structurally different so that they stop DNA synthesis when they are added to a strand

26. Reaction Mixture Copies of DNA to be sequenced Primer DNA polymerase
Standard nucleotides
Modified nucleotides

27. Reactions Proceed
Nucleotides are assembled to create complementary strands
When a modified nucleotide is included, synthesis stops
Result is millions of tagged copies of varying length

28. Recording the Sequence
T C C A T G G A C C
T C C A T G G A C
Recording the Sequence
T C C A T G G A
T C C A T G G
T C C A T G
T C C A T
T C C A
electrophoresis
gel
T C C
DNA is placed on gel
Fragments move off gel in size order; pass through laser beam
Color each fragment fluoresces is recorded on printout
T C
one of the many fragments of
DNA migrating
through the gel T
one of the DNA fragments
passing through a laser beam
after moving through the gel
T C C A T G G A C C A

29. Gene Libraries Bacteria that contain different cloned DNA fragments
Genomic library
cDNA library

30. Using a Probe to Find a Gene
You want to find which bacteria in a library contain a specific gene
Need a probe for that gene
A radioisotope-labeled piece of DNA
It will base-pair with the gene of interest

31. Use of a Probe Colonies on plate Cells adhere to filter
Cells are lysed;
DNA sticks
to filter
Probe is
added
Location where probe binds forms
dark spot on film, indicates colony with gene

32. Applications
What can genetic engineering be used for?

33. Engineered Proteins
Bacteria can be used to grow medically valuable proteins
Insulin, interferon, blood-clotting factors
Vaccines
Human gene is inserted into bacteria, which are then grown in huge vats

34. Cleaning Up the Environment
Microorganisms normally break down organic wastes and cycle materials
Some can be engineered to break down pollutants or to take up larger amounts of harmful materials
Break down oil, sponge up heavy metals

35. Basic Research Recombinant DNA technology allows researchers to:
Investigate basic genetic processes
Reconstruct life’s evolutionary history
Devise counterattacks against rapidly mutating pathogens

36. Engineered Plants Cotton plants that display resistance to herbicide
Aspen plants that produce less lignin and more cellulose
Tobacco plants that produce human proteins
Mustard plant cells that produce biodegradable plastic

37. First Engineered Mammals
Experimenters used mice with hormone deficiency that leads to dwarfism
Fertilized mouse eggs were injected with gene for rat growth hormone
Gene was integrated into mouse DNA
Engineered mice were 1-1/2 times larger than unmodified littermates

38. More Mouse Modifications
Experiments showed that human growth hormone genes can be expressed in mice
Human genes are inserted into mice to study molecular basis of genetic disorders, such as Alzheimer’s disease
Variety of methods used to introduce genes

39. Cloning Dolly 1997 - A sheep cloned from an adult cell
Nucleus from mammary gland cell was inserted into enucleated egg from another sheep
Embryo implanted into surrogate mother
Sheep is genetic replica of animal from which mammary cell was taken

40. Designer Cattle
Genetically identical cattle embryos can be grown in culture
Embryos can be genetically modified
Experimenters are attempting to create resistance to mad cow disease
Others are attempting to engineer cattle to produce human serum albumin for medical use

41. The Human Genome Initiative
Goal - Map the entire human genome
Initially thought by many to be a waste of resources
Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes
Sequencing was completed ahead of schedule in early 2001

42. Using Human Genes
Even with gene in hand it is difficult to manipulate it to advantage
Viruses usually used to insert genes into cultured human cells but procedure has problems
Very difficult to get modified genes to work where they should

43. Eugenic Engineering Selecting “desirable” human traits
Who decides what is desirable?
40 percent of Americans say gene therapy to make a child smarter or better looking would be OK

44. Where Do We Go Now?
Can we bring about beneficial changes without harming ourselves or the environment?
Gene therapy is not harmless
A young man died after gene therapy that used an adenovirus
Gene therapy can save lives
Infants with disabled immune systems are now healthy

45. Can Genetically Engineered Bacteria “Escape”?
Genetically engineered bacteria are designed so that they cannot survive outside lab
Genes are included that will be turned on in outside environment, triggering death

46. Effects of Engineered Organisms
Opposition to any modified organisms
What if engineered genes escape into other species?