This document discusses recombinant DNA technology and its various applications. Recombinant DNA is produced by splicing DNA from different sources for purposes such as cloning DNA, genetically engineering organisms, and producing proteins. Key aspects covered include restriction endonucleases, cloning strategies using vectors like plasmids and bacteriophages, cDNA and genomic libraries, DNA sequencing techniques like Sanger sequencing, PCR, expression of eukaryotic proteins in prokaryotes, microarrays, and gene silencing using siRNA.

1. RECOMBINANT DNA
METHODS AND
TECHNOLOGY
Matthew George, Jr., Ph.D.

2. Recombinant DNA and Genetic Engineering
 A recombinant DNA molecule is produced by splicing
together DNA from different sources. It is usually done for
one of the following reasons:
 To clone large amounts of DNA for the studies of gene
organization; structure; sequencing; and gene regulation
 To genetically engineer cells in an organism to confer new
desired characteristics (i.e., gene therapy, and correct
genetic defects)
 To produce the product(s) of spliced genes in bacterial cells
(i.e. insulin, human growth hormone)
 Alter the genetic characteristics of fruits and vegetables
 To produce organisms (i.e. Dolly, cows and mice for
therapeutic purposes)

STEM CELL RESEARCH: to produce new tissues and organs

4. Recombinant DNA Product Effect?

5. THE UTILITY OF RECOMBINANT DNA TECHNOLOGY

6. RESTRICTION ENDONUCLEASES

7. BASIC CLONING STRATEGY

8. EcoRI Restriction Endonuclease

9. EcoRI Recombinant

10. Vector Characteristics

11. Basic components of a plasmid cloning vector that
can replicate within an E. coli cell
Molecular Cell Biology, 7th Edition, Lodish et al.

12. CLONING VECTORS

13. Structure of pBR322 - a common cloning vector
• derived from a naturally occurring plasmid
•has unique restriction enzyme cleavage sites for
insertion of foreign DNA (100bp to 10kb)
• has antibiotic resistance genes for selection of
transformants containing the plasmid (insertion of DNA at these sites will
result in the bacteria becoming sensitive to that antibiotic)
•has origin of DNA replication (ori) for propagation in E. coli
Fig. 7.7

14. Yeast Artificial Chromosome (YAC)
Normal ends of
eukaryotic chromosomes

15. DNA cloning in a plasmid vector permits amplification of a DNA fragment*
See Devlin, fig. 7.9; *Molecular Cell Biology 7th Edition, Lodish et al.

17. Cloning a segment of DNA into a plasmid vect or
Human DNA cut with PstI PstI
P
ampR tetR pBR322 ampR, tetR
P P
combine
and
ligate P
tetR
pBR322 DNA cut with PstI
inactivating the amp R gene
tetR
pBR322 (human clone) tet R
• bacteria are “transformed” with the recombinant plasmid
• colonies that grow in tetracycline, but not in ampicillin are isolated

18. Antibiotic Selection
R

19. cDNA Formation

21. A cDNA library contains representative copies of cellular mRNA sequences*
See Devlin’s figure 7.16; *Molecular Cell Biology, 7th Edition, Lodish et al.

22. Genomic DNA Library

23. Lambda Phage Cloning

24. See fig. 7.13

26. Northern Blotting
(Transfer of RNA from a gel to a solid matrix)
Gives information about whether RNAs are present
in a tissue. Often used to determine whether
particular genes are expressed. Does NOT give
information about whether the gene is present in
other tissues.
Presence of Glucokinase
Human Liver Human Muscle Rat Brain E. coli
------

28. Expression Vectors
Fig. 7.22

29. Some eukaryotic proteins can be produced in E. coli cells
from plasmid vectors containing the lac promoter
Molecular Cell Biology, 7th Edition, Lodish et al.

30. Detection of Expression

32. PCR

39. The CSI Effect

41. Sanger Dideoxy Sequencing
Fig. 7.5

42. Dideoxynucleotide

45. Transgenic animals - the gene of interest together with appropriate regulatory elements is
microinjected into a fertilized mouse egg which is then inserted into the uterus of a foster
mother mouse

46. Microarray Technology

53. siRNA: “Gene Silencing”

54. Thank you for your time and
patience; make us proud and
good luck!!!