1. Biotechnology
Chapter 20

2. DNA Cloning Yields multiple copies of a gene or other dna segment
20.1

3. Biotechnology
Biotechnology – the manipulation of organisms or their components to make useful products
Biotechnology is used in all facets of life, and the advances being made in this field are only growing

4. DNA and Biotechnology
DNA is the molecule that holds the instructions for life
Scientists have learned how to manipulate it, and insert new pieces of DNA into pre-existing DNA
Recombinant DNA – DNA molecules formed when segments of DNA from two different sources are combined in vitro

5. DNA Cloning
Gene Cloning – The production of multiple copies of a single gene
Often to clone a gene, a bacteria is used
The plasmid of the bacteria is more easily manipulated, and a selected portion of DNA (or a gene) is inserted into this plasmid
The bacteria then reproduce asexually, producing identical copies of their DNA
Each new cell now has the desired gene
This can also be used to produce large quantities of a desired protein (not just the DNA)

6. Cloning Vectors Plasmids are also known as cloning vectors
Cloning Vector – a DNA molecule that can carry foreign DNA into a host cell and replicate there

7. Examples of Gene Cloning
Genes for pest resistance has been inserted into plants
Genes have been inserted into bacteria that allow them to break down oil spills
HGH has been produced using gene cloning
Proteins that have dissolve blood clots are produced using gene cloning

8. Restriction Enzymes Plasmid DNA is cut using restriction enzymes
Restriction Enzymes – an enzyme that cuts DNA at specific nucleotide sequences
In bacterial cells, restriction enzymes protect bacteria by cutting up DNA that is foreign to the cell so that it cannot infect the bacteria
Scientists have found restriction enzymes that will actually cut the bacterial plasmid, rather than foreign DNA

9. Restriction Enzymes
Restriction enzymes will cut up DNA in to many pieces because, by chance, the nucleotide sequence it is designed to cut will occur many times in a molecule
The resulting pieces are called Restriction Fragments
Effecting restriction enzymes will cut the sugar-phosphate backbone to have at least one single stranded end known as a sticky end
These sticky ends can be ‘glued’ into DNA using DNA Ligase

10. (Imagine below that this is an entire circular plasmid)
Cutting DNA
The DNA plasmid is first isolated
It is then cut by using restriction enzymes
(Imagine below that this is an entire circular plasmid) C T A G G C T A G C T T A A A A T T C G

11. The sticky ends allow outside pieces of DNA to be attached and inserted into the DNAG C T T A A A A T T C G
Sticky End

12. Transformation
Transformation – the change of a bacteria cell due to the uptake in incorporation of foreign DNA
Whenever a bacterial cell takes in new DNA it is said to have been ‘transformed’

13. Transforming Bacteria
The gene for HGH is removed from a human cell

14. The plasmid will have sticky ends in addition to the HGH gene
The plasmid is cut at certain sequences in the DNA using the same Restriction Enzyme used to cut the HGH gene from a human cell
The plasmid will have sticky ends in addition to the HGH gene

15. DNA Ligase covalently bond the sugar phosphate backbone
The gene for HGH is then mixed with the bacterial plasmid, and the HGH is incorporated into the plasmid
DNA Ligase covalently bond the sugar phosphate backbone
The plasmid is then added to the bacteria

16. This shows the process of transformation in bacteria
The bacteria now has the gene for HGH, and has the ability to produce it
This shows the process of transformation in bacteria

17. Markers
Often times, genes can be used as markers to make sure that the bacteria have undergone transformation:
FGP – Bacteria will glow under IR light if the gene was incorporated into the plasmid
AMP – Bacteria will be immune to the anti-biotic ampicillin

18. DNA Libraries
Genomic Library – a collection of DNA fragments that are stored and propagated in a population of cloning vectors
This allows researchers to study the entire genome of an organism

19. PCR
Polymerase Chain Reaction – A method used to amplify a piece of DNA quickly, many times, and without using cells
Beneficial if DNA segment is short
Often used in crime scenes

20. PCR - Process
DNA is incubated in a test tube with a special DNA Polymerase, nucleotides, and primers
DNA is heated to a certain temperature so that the DNA Ladder separates
It is then cooled, and the Polymerase and primers replicate the DNA
This cycle is repeated over and over, doubling the amount of DNA every cycle

21. 1. 1. 2. 1. 3. 2. 4. The number of DNA molecules doubles every cycle
The DNA multiplies exponentially 3. 2. 4.

22. DNA Technology allows us to study the sequence, expression, and function of a gene
20.2

23. Gel Electrophoresis
Gel Electrophoresis – Process that separates nucleic acids or proteins on the basis of charge, size, or other physical properties
Restriction enzymes are used to cut DNA up into different pieces
RFLP’s – ‘Restriction Fragment Length Polymorphisms’
Even in the same chromosomes of different organisms, there are differences that are seen in Restriction sites

24. DNA Mixed with different restriction enzymes are put into reservoirs on a gel electrophoresis pad
Negative
An electric current is then put into the gel. The current moves from the negative end to the positive end, and takes the different lengths of DNA with it +
Positive

25. The larger pieces of DNA don’t go as far, and the smaller pieces move farther and faster away from the original location
Negative
The bands that are formed can then be compared to other samples of DNA to eventually determine the genes in the DNA sequence +
Positive

26. Uses for Gel Electrophoresis
Identify different alleles based on DNA sequence
Determine whose DNA was found at a crime scene
Determine the father of a child

29. Reading the Sequence
Dyes are added to specific nucleotides and put into the test tubes
The dyes terminate DNA replication
Using gel electrophoresis, the different lengths of DNA are separated

30. T C G A T C G T G T
Dye Molecule

31. Genomics Study of entire genomes and how genes interact
Made possible through sequencing

33. DNA Microarray Assays Measure expression of thousands of genes at once
Compare gene expression of different cell types, stages of development, species, etc.

34. Proteomics Study of proteins made from genomes
Genetic complexity is determined by proteins produced not number of genes
Ex: Apples have 57,386 genes; humans have 20,000 genes
Apples produce 65,000 proteins; humans produce 100,000

35. Cloning organisms may lead to production of stem cells for research and other applications
20.3

36. Cloning Gene cloning (recombinant DNA) Reproductive Therapeutic
Generate an organism that has the same DNA as a current or previously existing animal
Mice, pigs, sheep, rabbits
Therapeutic
Embryo cloning for studying human development, stem cells

37. Safety & Ethical Questions
Just because we can does it mean we should?

38. The practical applications of dna technology affect our lives in many ways
20.4

39. Medical applications Diagnosis of disease Gene therapy
PCR, primers detect specific pathogens or genetic mutations that cause disease
Gene therapy
Insert normal alleles into someone whose cells contain defective alleles

40. Pharmaceutical Applications
Insulin, HGH manufactured by bacteria containing human gene
Pharmacogenomics
How your genes influence your body’s response to medications
Designer pharmaceuticals?

41. Forensics Uses combination of techniques to prove guilt/innocence
Small samples can be used for PCR, electrophoresis, Southern blotting, RFLP analysis

42. Environmental Cleanup
Bacteria can be modified to break down heavy metals, oil, and other toxins

43. Agricultural Applications
Animals
Manipulated to produce human proteins such as hormones, clotting factors
Manipulate to produce more of what we need, such as milk or eggs
Plants
Introduce pest resistance
Improve nutritional value (“golden” rice)