This document provides an introduction to biotechnology. It discusses key areas including cell technology such as isolating, growing, and fractionating cells. Molecular technology topics covered include DNA cloning, sequencing, gene expression, and analyzing protein functions. Specific techniques are described at a high level, such as fluorescence-activated cell sorting, cell culture, chromatography, electrophoresis, hybridization, PCR, and recombinant DNA technology. The goal of biotechnology is discussed as studying gene functions through approaches like genetic screening, monitoring expression, and genome manipulation.

1. Introduction to biotechnology
Haixu Tang
School of Informatics

2. Biotechnology
• Cell technology
– Isolating cells
– Growing cells in culture
– Fractionating cells
• Molecular technology
– DNA cloning
– DNA sequencing
– Gene expression
– Analyzing protein functions

3. Isolating Cells
• Disrupting extracellular matrix: proteolytic
enzymes or EDTA
• Separating different cell types
– Antibodies coupled to a fluorescent dye
(fluorescence-activated cell sorter)
– Microscopic dissection
• Cell growth

4. Fluorescence-activated cell sorter

5. Microscopic dissection

6. Growing cells in culture

7. Composition of a Typical Medium Suitable for
the Cultivation of Mammalian Cells
AMINO ACIDS VITAMINS SALTS MISCELLANEOUS PROTEINS
Arginine biotin NaCl glucose insulin
Cystine choline KCl penicillin transferrin
Glutamine folate NaH2PO4 streptomycin growth factors
Histidine nicotinamide NaHCO3 phenol red
Isoleucine pantothenate CaCl2 whole serum
Leucine pyridoxal MgCl2
Lysine thiamine
Methionine riboflavin
Phenylalanine
Threonine
Trytophan
Tyrosine
Valine

8. Cell line
• Most vertebrate cells stop dividing after a
finite number of cell divisions in culture –
senescence;
• "immortalized" cell line: telemerase
• Inactivate the checkpoint mechanisms
• Cell lines can often be most easily
generated from cancer cells.

9. Hybrid cells
• a heterocaryon, a combined cell with two
separate nuclei

11. Ultracentrifuge

12. Cell
fractionation by
centrifugation

13. velocity sedimentation vs.
equilibrium sedimentation

14. Column chromatography

15. Matrices used for chromatography

16. Protein
purification by
chromatography

17. SDS polyacrylamide-gel
electrophoresis

20. Western blotting

21. Protein identification via MS

22. DNA recombination technology
• 1. Cleavage of DNA at specific sites by restriction nucleases, which greatly
facilitates the isolation and manipulation of individual genes.
2. DNA cloning either through the use of cloning vectors or the polymerase
chain reaction, whereby a single DNA molecule can be copied to generate
many billions of identical molecules.
3. Nucleic acid hybridization, which makes it possible to find a specific
sequence of DNA or RNA with great accuracy and sensitivity on the basis of
its ability to bind a complementary nucleic acid sequence.
4. Rapid sequencing of all the nucleotides in a purified DNA fragment, which
makes it possible to identify genes and to deduce the amino acid sequence
of the proteins they encode.
5. Simultaneous monitoring of the expression level of each gene in a cell,
using nucleic acid microarrays that allow tens of thousands of hybridization
reactions to be performed simultaneously.

23. Restriction
nucleases

24. Restriction nucleases produce DNA
fragments that can be easily joined together

25. DNA Gel
electrophoresis

27. DNA hydridization

29. Gel transferred hybridization

30. In situ hydridization

31. In situ hybridization for RNA
localization

32. DNA recombination

33. DNA cloning

34. YAC

35. Human genome
library

36. The synthesis of cDNA

37. cDNA clones and genomic DNA
clones

38. PCR

39. PCR based gene cloning

40. PCR
used in
forensic
science

41. Protein
expression
system
via a plasmid
expression vector

42. Molecular biology research

43. Studying gene functions
• Genetic screening
• Monitoring gene expression
• Site-directed mutagenesis
• Gene replacement or knockout

44. LDA

45. Reporter gene

46. Site-directed Mutagenesis

47. Genome manipulation

48. Anti-sense RNA strategy

49. Making collections of mutant
organisms

51. Mouse with an engineered defect
in fibroblast growth factor 5
(FGF5)