1. Biotechnology The manipulation of organisms or their genes for
Basic biological research
Medical diagnostics
Medical treatment (gene therapy)
Pharmaceutical production
Forensics
Environmental clean up
Agricultural applications
Genetic components to make useful products

2. Biotechnology The genetic manipulation of organisms
Humans have been doing this for thousands of years
Plant & animal breeding

3. Evolution & breeding of food plants
Evolution in morphology of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties

4. Evolution of breeding of food plants
“descendants” of the wild mustard
Brassica spp.

5. Animal husbandry/breeding

6. Biotechnology today Genetic engineering Manipulation of DNA
Recombinant DNA
Nucleotide sequences from two different sources are combined in vitro into the same DNA molecule

7. Gene Cloning Preparing gene-sized pieces of DNA in identical copies
Materials needed: bacteria and their plasmids
Used for making copies of a particular gene and producing a protein product

8. Plasmids Small supplemental circles of DNA Carry extra genes
5000 – 20,000 base pairs
Self-replicating
Carry extra genes
2-30 genes
Can be exchanged
between bacteria
Rapid evolution
Antibiotic resistance
Can be imported from environment

9. Plasmids

10. Plasmids & antibiotic resistance
First recognized in 1950s in Japan
Bacterial dysentery not responding to antibiotics
Worldwide problem now

11. Genetic variation in bacteria can occur naturally through 3 different processes
Transformation – rare in nature
Giffith’s experiment
Transduction – bacteriophages carry bacterial genes from one bacterial cell to another
Conjugation – direct transfer of DNA between bacteria

12. Transduction
Phage viruses carry bacterial genes from one host to another

13. Conjugation
Direct transfer of DNA between 2 bacterial cells that are temporarily joined
E. coli “male” extends sex pilus, attaches to “female” bacterium
Cytoplasmic bridge allows transfer of DNA

14. Gene cloning Foreign DNA is inserted into a plasmid
Recombinant plasmid is inserted into a bacterial cell
Reproduction in the bacterial cell results in cloning of the recombinant plasmid
Permits production of multiple copies of a single gene
The original plasmid is called a cloning vector
DNA molecule that can carry foreign DNA into a host cell and replicate there

15. Transformation in the Laboratory
Transformation was first performed in the laboratory by Griffith and later by Avery, MacLeod, and McCarty (experiment using mice and pneumococcus bacteria – please review!)
Bacteria can take up DNA only during the period a the end of logarithmic growth – cells are said to be competent (can accept DNA that is introduced from another source)

16. E. coli competence can be induced under carefully controlled chemical growth conditions
Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells
E. coli cell membrane is weakened using ice cold CaCL2
E. coli cells are then “heat shocked” to induce them to take up the plasmid
Sterile technique must be used
Transformation Lab – we will transform bacteria by introducing a plasmid that will convey resistance to the antibiotic, ampicillin
Ampicillin kills bacteria by interfering with their ability to make cell walls

17. Figure 20.2 A preview of gene cloning and some uses of cloned genes
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2
Plasmid put into bacterial cell
Recombinant bacterium 3
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Copies of gene
Protein harvested
Figure 20.2 A preview of gene cloning and some uses of cloned genes 4
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth

19. “Cutting and Pasting” DNA to make Plasmids
Restriction enzymes (restriction endonucleases)
Cut DNA molecules at specific DNA sequences
Restriction sites
Many cuts yielding restriction fragments= RFLP (Restriction Fragment Length Polymorph)
Length depends upon the number of cuts made in a given piece of DNA and location of each recognition sequence
If DNA is cut in a staggered way, fragments with “sticky ends” that bond with complementary sticky ends of other fragments are produced
Symmetrical palindrome
The sequence is the same when one strand is read left to right and the other strand is read right to left

20. Paste DNA Sticky ends allow Ligase
H bonds between complementary bases to anneal
Ligase
Seals the bonds between restriction fragments
Joins sugar-phosphate bonds

21. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination 3
DNA ligase seals strands.
Recombinant DNA molecule

22. Restriction enzymes Discovered in the 1960s
In nature, bacteria use restriction enzymes for protection to cut foreign DNA (from invading viruses)
Named according to specific system
Letters refer to organism from which enzyme is isolated
Ex. EcoRI = 1st restriction enzyme found in E. coli
First letter = genus
Next two letters = species
Fourth letter = strain
Roman numeral = whether isolated first, second, etc.

23. Storing Cloned Genes in DNA Libraries
A genomic library, made using bacteria, is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
A genomic library, made using bacteriophages, is stored as a collection of phage clones
Each of these books is a clone of bacterial cells that contain copies of a particular foreign genome fragment in their recombinant plasmid

24. Making complementary DNA (cDNA)
Reverse transcription – can be used when cells produce large amounts of the particular polypeptide
cells will contain mRNA for polypeptide
mRNA is isolated and can be used to make the complementary DNA through reverse transcription – reverse of normal transcription
requires DNA nucleotides and enzymes called reverse transcriptases
after mRNA has been used to make DNA, mRNA is removed and the complementary strand of DNA is made by adding the enzyme, DNA polymerase and more DNA nucleotides
the result is a double-stranded DNA molecule identical to the original DNA molecule
reverse transcriptases were first obtained from retroviruses

25. Making cDNA for a eukaryotic gene
Reverse transcriptase is added to a test tube containing mRNA isolated from the cell
Reverse transcriptase makes the first DNA strand using RNA as a template and a stretch of thymine deoxyribonucleotides as a DNA primer

26. Making cDNA for a eukaryotic gene
mRNA is degraded by another enzyme
DNA polymerase synthesizes the second strand using a primer in the reaction system

27. Making cDNA for a eukaryotic gene
The result is cDNA which carries the complete coding sequence of the gene but not the introns

28. Expressing Cloned Eukaryotic Genes
After a gene has been cloned, its protein product can be produced in larger amounts for research
Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

29. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
Copying DNA without bacteria or plasmids
A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

30. PCR
Requires double-stranded DNA containing the target sequence, a heat-resistant DNA polymerase, all four nucleotides, and two 15 to 20 DNA nucleotides that serve as primers
Primers define section of DNA to be cloned
One primer is complementary to one end of the target sequence on one strand; the second primer is complementary to the other end of the sequence on the other strand

31. PCR Denaturation: Heat briefly to separate strands of DNA
Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence
Extension: DNA polymerase adds nucleotides to the 3’end of each primer

32. Kary Mullis- 1985 Development of PCR technique
A copying machine for DNA

33. DNA technology DNA cloning allows researchers to
Compare genes and alleles between individuals
Locate gene expression in a body
Determine the role of a gene in an organism
Several techniques are used to analyze the DNA of genes

34. DNA Fingerprinting – Uses
Also called DNA Profiling
Used to reveal a DNA pattern which is unique to an individual
Crimework: rape and murder cases (forensics)
Paternity suits
Missing persons and unidentified bodies
Immigration disputes
Animal work - breeding

35. Restriction Enzymes are used for DNA fingerprinting (profiling)
Creating a pattern of DNA bands on a gel
Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, a restriction enzyme will make many cuts
Result: production of fragments of DNA of various lengths – Restriction Fragment Length Polymorphs (RFLPs)
Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into different sized RFLPs

36. Electrophoresis means “to carry with an electric current”
The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern
Electrophoresis means “to carry with an electric current”
Different sized RFLPs will be carried different distances by an electric current as they migrate through an agarose gel inside a gel box
Electricity is run through the gel box creating a positive end and a negative end
Negatively charged DNA migrates from the negative end of the gel box through the pores in the gel to the positive end of the gel box
Smaller RFLPs will migrate farther than larger pieces, spreading the RFLPs across the gel in a bar-like pattern
Stain is used to make the DNA bands visible

37. SEM photo of a 1% LE Agarose gel at 22kX magnification

38. Measuring fragment size
Compare bands to known “standard”
Usually lambda phage cut with HindIII
Nice range of size with a distinct pattern

39. Genomes
Complete genome sequences exist for a human, chimpanzee, E. coli, brewer’s yeast, nematode, fruit fly, house mouse, rhesus macaque, and other organisms
Comparisons of genomes among organisms provide information about the evolutionary history of genes and taxonomic groups
Genomics
the study of whole sets of genes and their interactions
Bioinformatics
the application of computational methods to the storage and analysis of biological data
The Human Genome Project established databases and refined analytical software to make data available on the Internet
This has accelerated progress in DNA sequence analysis

40. Centralized Resources for Analyzing Genome Sequences
Bioinformatics resources are provided by a number of sources:
National Center for Biotechnology Information (NCBI)
Created by the National Library of Medicine and the National Institutes of Health (NIH) European Molecular Biology Laboratory
Genbank, the NCBI database of sequences, doubles its data approximately every 18 months
Online visitors can search for matches to
A specific DNA sequence
A predicted protein sequence
Common stretches of amino acids in a protein
The NCBI website also provides 3-D views of all protein structures that have been determined
DNA Data Bank of Japan
European Molecular Biology Laboratory

41. Fig. 21-4
Figure 21.4 Bioinformatics tools available on the Internet