1. Biotechnology
CHAPTER 20

2. History
1995 – first time the entire genome of an organism was sequenced; prokaryote - Haemophilius influenzae
2007 – Researchers had sequenced hundreds of prokaryotic genomes and dozens of eukaryotic ones
Among the eukaryotic genomes was the human genome; all 3 billion base pairs (Human Genome Project)

3. Definitions Biotechnology Genetic Engineering
The manipulation of organisms and their components to make useful products
Genetic Engineering
The direct manipulation of genes for practical purposes
NOT PRACTICAL!

4. We are going to break this chapter up into 5 parts:
Techniques for manipulating DNA
Analyzing gene sequences
Analyzing gene expression
Cloning and stem cells
Practical applications of biotechnology

5. DNA Manipulation Techniques
Recombinant DNA
Cloning Genes
Storing Cloned Genes
Cloned Gene Identification (Nucleic Acid Hybridization)
Amplifying DNA (PCR technique)

6. DNA MANIPULATION TECHNIQUES – 1. Recombinant DNA
DNA molecules formed when segments of DNA from two different sources – often different species – are combined in vitro (in a test tube)

7. Making Recombinant DNA
DNA MANIPULATION TECHNIQUES – 1. Recombinant DNA
Making Recombinant DNA
Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific
Restriction site: recognition sequence for a particular restriction enzyme
Restriction fragments: segments of DNA cut by restriction enzymes
Sticky end: short extensions of restriction fragments
DNA ligase: enzyme that can join the sticky ends of DNA fragments
Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

8. DNA MANIPULATION TECHNIQUES – 2. Cloning Genes
We typically use bacteria to clone genes (make extra copies)
Why?
Bacteria have plasmids – small circular DNA molecules that replicate separately from the single, circular chromosome
Plasmids can be removed and used to make recombinant DNA
The recombinant plasmids can then be reinserted back into the bacterial cell (bacterial transformation)
Cloning vector – any DNA molecule that can carry foreign DNA into a host cell and replicate there
Bacteria multiply quickly thus making thousands of copies of the gene in under 24 hours!

9. DNA MANIPULATION TECHNIQUES – 2. Cloning Genes
Single-Gene Cloning
Make recombinant DNA using a bacterial plasmid (cloning vector) and the gene of interest
Transform bacteria by inserting the recombinant plasmid into a bacteria cell
When the bacteria cell divides, it will copy both the recombinant plasmid and its circular chromosome
Each descendent of the original transformed cell will have a copy of the gene
How do you know if the process worked?
The gene will be expressed by the bacteria (it will produce the protein the gene codes for)

10. Gene Cloning: Shotgun Approach
DNA MANIPULATION TECHNIQUES – 2. Cloning Genes
Gene Cloning: Shotgun Approach
Many times scientists will cut up an entire genome without targeting a single specific gene
This results in many different recombinant plasmids as each restriction fragment (gene) can be used to make a recombinant plasmid (one fragment – one plasmid)
Then each recombinant plasmid can be inserted into different bacterial cells
Each transformed bacteria cell can clone itself millions of times forming a visible colony of bacteria that are all genetically identical

11. DNA MANIPULATION TECHNIQUES – 3. Storing Cloned Genes
All of the colonies together on a single plate is called a genomic library
There are many types of genomic libraries and they differ based on the cloning vector
Examples
Plasmid library
Phage library
Bacterial artificial chromosome library (BAC – basically a giant plasmid)
Each colony can be stored in separate wells on a “multiwell” plastic plate

12. 4. Cloned Gene Identification
DNA MANIPULATION TECHNIQUES – 4. Cloned Gene Identification
4. Cloned Gene Identification
Now that you have seen the shotgun approach to gene cloning and how the cloned genes are stored, there is one small problem…
How do you know which gene was cloned in each colony?
You would need to know at least one of the following:
Part of the nucleotide sequence of the gene
Part of the nucleotide sequence of the gene in a closely related species
The amino acid sequence of the protein the gene codes for

13. Complimentary Nucleic Acid Probes
DNA MANIPULATION TECHNIQUES – 4. Cloned Gene Identification
Complimentary Nucleic Acid Probes
Knowing part of the sequence allows you to create a short, single-stranded nucleic acid (nucleic acid probe) that is complimentary to the nucleotide sequence you know from the gene
You can synthesize the probe using nucleotides labeled with radioactive isotopes for easy identification
Example)
Gene sequence you know: ’-CTTAGGTCA-3’
Complimentary probe sequence: ’-GAATCCAGT-5’
The probes can be used for nucleic acid hybridization

14. Nucleic Acid Hybridization
DNA MANIPULATION TECHNIQUES – 4. Cloned Gene Identification
Nucleic Acid Hybridization
Using the multiwell genomic library plate, transfer a few clones from each well to a documented spot on a special nylon membrane
The nylon membrane contains chemicals that will break open the cloned cells and denature their DNA; the resulting single-stranded DNA molecules stick to the membrane
The membrane is then bathed with the nucleic acid probes. The probes base pair with the gene of interest
Excess DNA is washed off
A photographic film is laid over the membrane and any radioactive areas will become visible on the film
All spots can be traced back to the well in which the DNA came from 1 2 4

15. DNA MANIPULATION TECHNIQUES – 5. Amplifying DNA
DNA cloning in cells remains the best method for preparing large quantities of a particular gene or other DNA sequence
But what if the source of DNA is damaged/incomplete, contaminated, or impure?
Use a technique called polymerase chain reaction (PCR)

16. DNA MANIPULATION TECHNIQUES – 5. Amplifying DNA
Advantages of PCR
It’s FAST - billions of copies of a gene of interest or a DNA sequence can be made in a matter of hours!
It’s highly selective – only minute amounts of DNA are needed
The DNA can be damaged but as long as there are a few molecules containing the complete sequence you are interested in, it will work
Applications include ancient DNA analysis, crime scene analysis (DNA fingerprinting), prenatal genetic disorder testing

17. DNA MANIPULATION TECHNIQUES – 5. Amplifying DNA
Disadvantages of PCR
It’s not error proof and there is no spell check – proves why cloning genes in cells is better when large quantities are needed
All copies made using PCR must be sequenced to determine if they are correctly made (time consuming)

18. PCR Procedure A machine heats the DNA to separate the strands
DNA MANIPULATION TECHNIQUES – 5. Amplifying DNA
PCR Procedure
Virtual Lab
A machine heats the DNA to separate the strands
A primer, specific for the sequence you want to copy, is made, and added, so DNA polymerase can attach
New nucleotides are added to the 3’ ends until each strand is complete
The two strands go through the cycle again producing 4 strands
At the end of the 3rd cycle there will be 8 strands and 2 of them will match the target sequence exactly
As the process above is repeated, the number of copies increases exponentially!

19. Analyzing Gene Sequences
Gel Electrophoresis
Southern Blotting
DNA Sequencing (Sanger Method)

20. ANALYZING GENE SEQUENCES – 1. Gel Electrophoresis
DNA samples from different sources are cut using the same restriction enzymes
The mixture of DNA fragments for each source is placed into a “well” on an agarose gel
Electricity is passed through the gel to separate the DNA fragments. DNA is negatively charged so it will move toward the positive end of an electrophoresis chamber
The smaller the fragment, the farther it travels down the gel
Virtual Lab

21. Applications of Gel Electrophoresis
ANALYZING GENE SEQUENCES – 1. Gel Electrophoresis
Applications of Gel Electrophoresis
Restriction Fragment Analysis
Allows for comparison of two or more DNA molecules
Example
Comparing 2 alleles of a gene
A restriction enzyme recognizes a specific sequence of nucleotides
A change in just one nucleotide pair in the restriction sequence stops the restriction enzyme from cutting
This changes the number and size of restriction fragments produced

22. 2. Southern Blotting Problem
ANALYZING GENE SEQUENCES – 2. Southern Blotting
2. Southern Blotting
Problem
The restriction fragment analysis of two alleles requires the use of purified alleles
Using large genomes such as ours produces SO MANY fragments that the electrophoresis gel looks like one long smear from one end to the other
Southern Blotting technique allows us to get around this problem and focus on just the fragments of the gene of interest
Combines gel electrophoresis with nucleic acid hybridization

23. Southern Blotting Procedure
ANALYZING GENE SEQUENCES – 2. Southern Blotting
Southern Blotting Procedure
Cut the DNA samples using the same restriction enzymes
Separate the fragments using gel electrophoresis
Stack the following items from the bottom up:
Sponge soaked in an alkaline solution; agarose gel; nylon membrane; paper towels; heavy weight
Paper towels on the nylon membrane will pull the alkaline solution through the gel thus transferring the DNA to the nylon membrane
Nylon membrane denatures the DNA fragments
Bathe the nylon membrane with radioactive probes synthesized to be complimentary to the gene fragments of interest
Apply a photographic film to locate the bands containing DNA that base-paired with the probes

25. ANALYZING GENE SEQUENCES – 3. DNA Sequencing
Once a gene is cloned, its complete nucleotide sequence can be determined
This is done by sequencing machines modeling a method called the dideoxyribonucleotide chain termination method (aka the dideoxy method or the sanger method because he developed it)
Application:
Knowing a gene’s sequence allows us to compare it to similar genes in other species, where the function of the gene product may be known – if the genes are similar then we can assume their products have similar functions

26. Sanger Method Procedure
ANALYZING GENE SEQUENCES – 3. DNA Sequencing
Sanger Method Procedure
The DNA fragment with unknown base sequence is denatured and placed in a test tube with a primer, DNA polymerase, and the four nucleotides (A, T, G, C) in which some of the bases have been marked with a chemical dye
You must know part of the nucleotide sequence in order to make a primer.
The primer attaches to the 3’ end and DNA polymerase begins to replicate the DNA fragment; if a base with a chemical dye is added, the process stops and the incomplete fragment is removed
The replication process is repeated over and over again producing many different length fragments
The fragments are separated using gel electrophoresis and the sequence is determined using a fluorescence detector