1. Recombinant DNA Technology (genetic engineering)
Chapter 3
Recombinant DNA Technology
(genetic engineering)

2. Enzymes that cut and paste DNA
Restriction enzymes cut DNA at specific
base sequences called restriction sites

3. Enzymes that cut DNA are called restriction enzymes

4. Enzyme DNA ligase enzyme pastes cut ends back together

5. Cloning: the introduction of new or foreign genes into plasmids and other “vectors”
This is when scientists take control of the natural processes that the bacteria have evolved to promote exchange of genes between individuals of the same or different “species”
Circular extrachromosomal DNA found commonly in bacteria
Plasmid DNA is replicated at same time chromosomal DNA is replicated
Used to pass genes back and forth between different bacteria A T C G

6. Bacterial cells are efficient ways to produce lots of copies of a foreign gene introduced into a plasmid

7. Cloning Plasmids serve as cloning vectors
“T”umor-”i”nducing
DNA (Ti plasmid)
contains 8 tumor-
inducing genes
transformation
Use this plasmid to introduce a new gene into a plant chromosome

8. Concerns about cloning
What might happen if cloned bacteria were to leave the lab and transfer their genes to other bacteria or even humans?
E. coli was initially the most common host for these cloned genes
Benefits and hazards discussed in 1975 at a meeting
National Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC)
Guidelines established for recombinant DNA research by scientific community

9. Review of molecular biologists’ toolbox
Plasmids
Restriction enzymes
DNA ligase
Host bacterial cells to replicate plasmids

10. Recombinant DNA technology has become a way for geneticists to express genes from other organisms in bacteria
Human insulin gene was cloned into a bacterial plasmid
and expressed (gene mRNA protein) in a bacterium in 1977.
Cheap and pure source of insulin
Humulin growth hormone was first recombinant
DNA product to be approved by FDA in 1992
Currently over 100 products on market produced by
recombinant techniques

11. Mix plasmid and foreign DNA together with restriction enzyme and DNA ligase
This plasmid has the lacZ gene inserted
Section of foreign DNA with gene of interest
Multiple cloning site inside lacZ gene (restriction site for insertion site for
foreign gene) P O
Plasmid cloning vector
Foreign DNA
Restriction site
Restriction enzyme
DNA ligase

12. Plasmid cloning vector
Extra-chromosomal DNA
carried by bacterial cell
Ampicillin resistance gene ampR
(selective marker)
Multiple cloning site inside lacZ gene
(restriction site for insertion site for
foreign gene)
Foreign gene inserted
lacZ gene with promoter (used to switch in expression of foreign gene when inside a bacterial host cell)

13. Insert plasmid into host bacterial cell for replication
chromosome

14. Cultivate host cell to replicate and produce many copies of foreign gene
Bacterial cell

15. Detecting cells that have foreign gene inserted in lacZ gene on plasmid
Need some way to check to see that foreign gene was inserted into the plasmid so when you cultivate the cell, you know you are producing more copies of foreign gene

16. Switching on expression of foreign gene during cultivation of host bacterial cell
plasmid
No foreign gene inserted
RNA polymerase
chromosome
mRNA
enzyme
colored product
If no foreign gene inserted into restriction site, then blue colored product is produced
xGal (lactose)

17. If foreign gene is inserted into restriction site, then no colored product is produced
RNA polymerase
mRNA
no enzyme
xGal
no product (no color)

18. Plating cells on agar surface to promote colony formation
Visible colony of identical cells
Medium contains ampicillin to allow only the bacterial cells that contain plasmid with ampR gene to grow
Semisolid nutrient medium for bacterial cell to replicate to produce many daughter cells to form a visible colony

19. Cloning
(restriction sites)

21. Types of vectors 6-12 25 35 300 200-1000 Bacterial plasmid
Maximum insert size
(kilobases or kb [1000bp])
Bacterial plasmid
bacteriophage
cosmids
bacterial artificial chromosome
yeast artificial chromosome
6-12 25 35
300

22. Practical Features of DNA Cloning Vectors (Plasmids)
origin of replication (ori)
multiple cloning sites (MCS) or restriction sites
selectable markers
RNA polymerase promoter sequences
DNA sequencing primer sequences
Allows bacteria with this plasmid to grow in presence of ampicillin antibiotic
ori
ampR
lacZ gene
MCS
If plasmid picks up a foreign piece of DNA at the MCS, then the lacZ gene is non-functional

23. You can use plasmids to create a clone “library”
Purpose: To distribute different sections of a DNA molecule or chromosome into a vector that allows the genes contained in the section to be characterized

24. Making a genomic library
Plate out to
form colonies

25. Screening clones for plasmids that picked up foreign DNA fragment

26. What if you know a part of the base sequence of the gene you are looking for?
The Human Genome Project has given us this information for all the genes in our chromosomes

27. stopped

28. Polymerase chain reaction (PCR)
Has revolutionized molecular biology and biotechnology.
Most useful when you know at least some of the base sequence of the gene you are interested in
Only need to know a sequence containing base pairs in a gene that may contain thousands of base pairs

29. Design primers that specifically target sequences at the ends of the foreign gene
Plasmid A C G T
3’ 5’
5’ 3’
DNA polymerase
Forward primer
Reverse primer
Foreign gene

30. Polymerase Chain Reaction (PCR)
Much more rapid
approach to cloning
than making or
screening clone
libraries.
Makes lots of copies
of foreign gene
that is then inserted
into plasmid
Need to know part of sequence of gene

31. Cloning a gene by PCR Uses a restriction enzyme
that recognizes A-T restriction
site for cutting T vector for
insertion of gene

32. T-plasmid vector containing
same foreign gene
Host bacterial cell

33. Now, every “transformed”
bacterial cell that picks up
the plasmid contains the
same fragment “gene” of
foreign DNA

34. How do you recover foreign DNA fragment containing gene of interest?
Pellet cells from culture medium
Resuspend cells in solution that breaks up “lyses” cells to release DNA
Separate host cell DNA from plasmid DNA by electrophoresis
DNA bands

35. Separating DNA fragments produced by treatment with restriction enzymes
Agarose gel electrophoresis

36. Restriction mapping
Each band represents a different size fragment created by cutting the chromosome with a restriction enzyme Different lanes on gel contain fragments of same DNA cut with different restriction enzymes When you separate DNA fragments on a gel it is called a Southern gel

37. Restriction Mapping
This is the technique
used for DNA fingerprinting
Fragment of
chromosome

38. Gels that show genes that are being expressed
Gels that reveal mRNA
or other types of RNA
are called Northern gels

39. Testing all genes expressed in a tissue quickly using microarray or “gene chip”

40. short, single-stranded DNA-a different gene in each spot
Each spot contains
millions of copies of
short, single-stranded
DNA-a different gene in each spot
ACCTC
UGGAG
AACTC

41. Computer scans chip and provides a printout of which genes were expressed

42. Bioinformatics Database manipulation of DNA sequence information
Application of computer science and information technology to help understand biological processes
Use of computers to relate gene sequence to protein structure and function

43. Example of bioinformatics
Alignment of overlapping sequences
used to assemble sequence of large pieces of DNA (chromosomes)

44. Using Bioinformatics
GenBank-a library of base sequences that have been catalogued
useful for matching your sequences from your clone library with sequences found and deposited by others previously
go to blastn
type in AATAAGAACCAGGAGTGGA
BLAST finds the match to your sequence to be the gene for early-onset breast cancer, BRCA-1
each unique sequence is assigned an accession number to make it easy for scientists to refer back to that sequence

45. Comparing the human and mouse genome

46. More things you can do www.ncbi.nlm.nih.gov/Omim search Omim database
type in a word for a disease then search
the database provides you with a list of diabetes-related genes
click on one-it provides you with all types of information on these genes
click on gene map
click on IDDM1
click on 6p21.3
it shows you the locus on the chromosome where the gene resides (find )
click on it verifies that you have located the gene of interest

47. Search for a gene you are interested in
lists different metabolism along left
at top “click here” takes you to all the chromosomes
click on chromosome 7
gives you more info on the genes on that chromosome
shows you where the genes for different diseases are located on that chromosome.

48. Summary Restriction sites and enzymes Cloning vectors (plasmids)
Inserting foreign genes in plasmids
Hosts cells for replicating plasmids (bacteria)
Clone libraries, cDNA libraries
Screening for recombinant plasmids
Polymerase chain reaction (PCR)
Reverse transcription PCR for detecting mRNA
Separating DNA fragments on gels
Gene chips
Bioinformatics

49. Some companies doing this work want to patent the sequences of fragments of our DNA
A patent gives legal exclusive right to control use
of sequences contained within fragment
They see opportunities to turn
this into a money-making endeavor
Cost of bringing a new drug (protein) to market is about $500 million
Takes 5-8 years to do this

50. Patents
Companies want assurances that after investing their resources to get a product approved for use that another company can’t come in and make $$ without such an investment
Since 1980, the U.S. Patent Office has awarded patents on more than 20,000 gene sequences

51. Patent process 3 categories
products or composition of matter
methods of use
manufacturing processes
Conditions that must be met to receive a patent
must be new (not previously published or described)
must be useful
not obvious to one skilled in the field

52. Elements of a patent application
Description of technical field to which invention applies
Description of problems to be solved and prior “art”
How the invention improves upon prior art
Summary enumerating fundamental components of invention
Description of invention and indispensable steps for constructing invention

53. Elements of a patent application
Claims that outline the elements to be protected by law.
A claim cannot be so broad that it infringes upon prior “art”
A claim should not be so narrowly focused that the applicant could risk losing property claims
Patent attorneys are skilled in preparing patent application
Upon review of application by U.S. Patent Office, a decision will be made whether a new patent is justified-if so, a patent no. is assigned

54. New Patent Issues Sequences may or may not encode a gene
Sequence may control regulation of nearby genes.
Many scientists believe patenting should be reserved for the new technology used to discover genes and their functions and their application rather than the sequence.
Is it ethical to patent a sequence?
What are the possible consequences?