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Working with DNA: Isolation and Fingerprinting. Funding and support received from…. Today’s Agenda:. Introduction Safety Basic Practice “Using a Pipetteman” DNA Isolation Procedures Restriction Enzymes and Gels Yellowstone National Park and Bacterial Mats
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Today’s Agenda: • Introduction • Safety • Basic Practice “Using a Pipetteman” • DNA Isolation Procedures • Restriction Enzymes and Gels • Yellowstone National Park and Bacterial Mats • Practice DNA Fingerprinting Problems • Analysis of our Fingerprinting Gels • Bacteria and DNA Basics • Closing
Our Research Project - What We Are Cloning and Why • We hope to identify new hot spring bacteria that cannot be grown on lab media • To study these organisms, we extract DNA from hot springs that contain unknown bacteria
Our Research Project - What We Are Cloning and Why • We clone a specific identification gene (the 16S gene) from the hot spring DNA • We place each hot spring gene into E. coli, our cloning factory • And then we fingerprint and DNA sequence each hot spring clone
Words to the cautious… Neither the E. coli we use nor the hot spring bacteria we study have ever been shown to be pathogenic. Although you will be working with E. coli, you will never come in contact with hot spring bacteria… just their DNA after it has been extracted from the once-living cells.
Introduction: • All living things contain cells • Eukaryotes: more than one cell • Prokaryotes: one cell organisms
Bacteria come in many different shapes and sizes…take a quick look…
Bacteria can replicate easily… • To grow, bacteria divide and divide and divide again. • Problem: If you started with only 1 bacterial cell, and it divided 10 times, how many bacteria would you then have??
Bacteria are everywhere… Don’t panic!! This is a good thing. We have bacteria growing on our bodies which are supposed to be there.
What are Bacteria? • Bacteria are prokaryotes, meaning they are only one celled organisms. They are very small and can be harmful or beneficial.
Plasmids can also be found in bacterial cells: • Plasmids are: Mini-chromosomes found only in some bacteria • (1,000-10,000 base pairs) • Free-floating in the cytoplasm - not membrane-bound like chromosome • Naturally carry many antibiotic resistance genes • Replicate on their own
Plasmids and Cloning • Bacteria are used in genetic engineering and cloning because they serve as the factories for expressing foreign genes like insulin. Without plasmids, there would be no way to clone and express foreign genes.
Now we are going to do some work!!! DNA Precipitation
What are we using now? • 3M Sodium acetate: contributes ions to bind with positive phosphates open on DNA • Isopropanol: polar solution which attaches to DNA for precipitation • - 80C Freezer: Speeds the precipitation reaction with low amounts of DNA
What do we know about DNA? • Structure: Composed of nucleotides (monomer) consisting of: 1) phosphate group 2) deoxyribose sugar 3) one of four nitrogen bases
What do we know about DNA? • Structure: Nitrogen bases are named: - adenine (A) - guanine (G) - thymine (T) - cytosine (C)
What do we know about DNA? • Structure: • The structure of these nucleotides determines how they fit together. • Adenine fits with Thymine • Guanine fits with Cytosine
What do we know about DNA? • Structure: • DNA is “double-stranded” • The nucleotides are linked together covalently • Phosphate – Sugar – Phosphate – Sugar etc. • This is the “backbone”
What do we know about DNA? • Structure: • The two strands are oriented in opposite directions • The two strands are wound around each other forming the “helix” structure
What do we know about DNA? • Function: • Codes for 80,000 genes, which form proteins…the building blocks of life.
EukaryoticDeoxyribonucleic Acid • DNA for Short • Double helix - two strands made up of A, T, G, and C bases • Complex organisms - many linearchromosomes (10,000,000,000 or more base pairs)
ProkaryoticDeoxyribonucleic Acid • Bacteria - one circular chromosome (1,000,000 base pairs) • Chromosomes, in both cases, are held by proteins to the cell or nuclear membrane • Most RNA is translated into proteins that have structural or functional jobs in cells
Let’s get our samples now and continue on with our isolation… • Centrifuge: Spins solution at high speed to concentrate DNA at the bottom • TE: buffer at pH 8.0 • RNAse: enzyme which removes RNA present in sample through digestion
Restriction Enzymes • Cut specific sequences of DNA • Many different kinds • Named after organism they came from, enzyme number • E.g. EcoR1
Bacteria Produce Restriction Enzymes • Uniquely bacterial protection mechanism…why? • Restriction enzymes are short nucleotide sequences isolated from bacteria cells that protect them from virus.
Bacteria Produce Restriction Enzymes • When a viral DNA enters the bacterial cell, the restriction enzyme is able to recognize a specific sequence (restriction site) on the DNA molecule, which is usually 4-8 nucleotides long. The restriction enzyme will cut the viral DNA at these sites and hence restrict the growth of the virus.
Bacteria Produce Restriction Enzymes • Several hundreds of these enzymes have been isolated from various organisms and most are available commercially. These enzymes are used to cut a segment of gene from a human DNA molecule.
DNA Fingerprinting • DNA fragments are separated using gel electrophoresis • Each band represents the DNA which has been cut into smaller pieces using restriction enzymes
Gel Electrophoresis From your studies of DNA, can you tell me what charge DNA has?
Gel Electrophoresis • Gel is made of water and agarose • Wells on one end are where gels will be loaded with our samples
Gel Electrophoresis • The gel box contains water and buffer to keep the pH constant • Gel box has platinum wire that conducts protons and electrons • Gel box will be wired to the power source following the load
Gel Electrophoresis • To the strand of DNA moving through the agarose, the gel looks like a big mesh-like maze • The DNA travels through the maze as fast as it’s size will allow
Gel Electrophoresis • DNA moves from the negative towards the positive • Smaller – faster • Larger – slower • Where will these three end?
Gel Electrophoresis • Review: ** DNA travels – to + ** When the power supply turns off, we can see where the bands are and infer which are bigger and smaller ** Small goes far ** Large goes not far
DNA Fingerprinting Questions and Answers Do you know the answers to these questions?
DNA Fingerprinting • How good (accurate) is it at identification. For example, is it as good as classical fingerprints?
Question 1: • How good (accurate) is it at identification. For example, is it as good as classical fingerprints? • Answer: In theory, with the exception of identical twins, EVERYONE on this planet has a different DNA fingerprint. That is, DNA fingerprinting IS as good (distinctive/unique/specific) as classical fingerprinting for identification.
Question 2: • What are its advantages? • Answer: In theory DNA fingerprinting will work with much smaller amounts of material than a classical fingerprint & DNA lasts much longer than classical fingerprints. DNA-containing samples that are many years old (up to 25 million yr.) are still usable. Only very tiny quantities of DNA are required in order to carry out a highly accurate test. For example, dried blood, semen, spit, skin etc. on samples stored in dusty files for years are still usable. Samples of mixed DNA's can also be used. DNA containing evidence is much harder to clean up at a crime scene than other evidence, like classical fingerprints.
