Working with DNA: Isolation and Fingerprinting. Funding and support received from…

Working with DNA: Working with DNA: Isolation and Isolation and

Fingerprinting Fingerprinting

Funding and support Funding and support received from…received from…

Today’s Agenda:Today’s Agenda:

1)1) IntroductionIntroduction2)2) SafetySafety3)3) Basic Practice “Using a Pipetteman”Basic Practice “Using a Pipetteman”4)4) DNA Isolation ProceduresDNA Isolation Procedures5)5) Restriction Enzymes and GelsRestriction Enzymes and Gels6)6) Yellowstone National Park and Bacterial MatsYellowstone National Park and Bacterial Mats7)7) Practice DNA Fingerprinting ProblemsPractice DNA Fingerprinting Problems8)8) Analysis of our Fingerprinting GelsAnalysis of our Fingerprinting Gels9)9) Bacteria and DNA BasicsBacteria and DNA Basics10)10)ClosingClosing

Our Research Project - Our Research Project - What We Are Cloning and What We Are Cloning and WhyWhy• We hope to identify We hope to identify

new hot spring new hot spring bacteria that cannot bacteria that cannot be grown on lab be grown on lab media media

• To study these To study these organisms, we organisms, we extract DNA from extract DNA from hot springs that hot springs that contain unknown contain unknown bacteriabacteria

Our Research Project - Our Research Project - What We Are Cloning and What We Are Cloning and WhyWhy• We clone a specific We clone a specific

identification gene (the identification gene (the 16S gene) from the hot 16S gene) from the hot spring DNA spring DNA

• We place each hot We place each hot spring gene into E. coli, spring gene into E. coli, our cloning factoryour cloning factory

• And then we fingerprint And then we fingerprint and DNA sequence each and DNA sequence each hot spring clonehot spring clone

Words to the cautious…Words to the cautious…

Neither the E. coli we use nor Neither the E. coli we use nor the hot spring bacteria we the hot spring bacteria we

study have ever been shown to study have ever been shown to be pathogenic. Although you be pathogenic. Although you

will be working with E. coli, you will be working with E. coli, you will never come in contact with will never come in contact with hot spring bacteria… just their hot spring bacteria… just their DNA after it has been extracted DNA after it has been extracted

from the once-living cells.from the once-living cells.

Introduction:Introduction:

• All living things contain cellsAll living things contain cells

• Eukaryotes: more than one cellEukaryotes: more than one cell

• Prokaryotes: one cell organismsProkaryotes: one cell organisms

The Boring (Yawn!!) The Boring (Yawn!!) Eukaryotic Plant and Eukaryotic Plant and Animal Cells…Animal Cells…

http://www.microscopy.fsu.edu/cells/plants/plantmodel.html

http://www.microscopy.fsu.edu/cells/animals/animalmodel.html

The Exciting Bacterial The Exciting Bacterial Cell…Cell…

Bacteria come in many Bacteria come in many different shapes and sizes…different shapes and sizes…take a quick look…take a quick look…

Bacteria can replicate Bacteria can replicate easily…easily…

•To grow, bacteria To grow, bacteria dividedivide and and

divide divide and and dividedivide again. again.

•Problem: If you started with only Problem: If you started with only 1 bacterial cell, and it divided 10 1 bacterial cell, and it divided 10 times, how many bacteria would times, how many bacteria would you then have??you then have??

Bacteria are everywhere…Bacteria are everywhere…

Don’t panic!!Don’t panic!! This is a good thing.This is a good thing.

We have bacteria growing on We have bacteria growing on our bodies which are our bodies which are

supposed to be there. supposed to be there.

What are Bacteria?What are Bacteria?

• Bacteria are prokaryotes, meaning they Bacteria are prokaryotes, meaning they are only one celled organisms. They are are only one celled organisms. They are very small and can be harmful or very small and can be harmful or beneficial. beneficial.

Bacteria can cause diseases, Bacteria can cause diseases, like we all know…like we all know…

Bacteria can also have Bacteria can also have beneficial uses…beneficial uses…

Bacterial Cell Bacterial Cell Components…Components…

Plasmids can also be found Plasmids can also be found in bacterial cells:in bacterial cells:• Plasmids are: Mini-Plasmids are: Mini-

chromosomes found chromosomes found onlyonly in some bacteria in some bacteria

• (1,000-10,000 base (1,000-10,000 base pairs)pairs)

• Free-floating in the Free-floating in the cytoplasm - not cytoplasm - not membrane-bound like membrane-bound like chromosomechromosome

• Naturally carry many Naturally carry many antibiotic resistance antibiotic resistance genesgenes

• Replicate on their ownReplicate on their own

Plasmids and CloningPlasmids and Cloning

• Bacteria are used in genetic Bacteria are used in genetic engineering and cloning because engineering and cloning because they serve as the factories for they serve as the factories for expressing foreign genes like insulin. expressing foreign genes like insulin. Without plasmids, there would be no Without plasmids, there would be no way to clone and express foreign way to clone and express foreign genes. genes.

Now we are going to do Now we are going to do some work!!!some work!!!

DNA PrecipitationDNA Precipitation

What are we using now?What are we using now?• 3M Sodium acetate: contributes ions 3M Sodium acetate: contributes ions

to bind with positive phosphates open to bind with positive phosphates open on DNAon DNA

• Isopropanol: polar solution which Isopropanol: polar solution which attaches to DNA for precipitationattaches to DNA for precipitation

• - 80C Freezer: Speeds the - 80C Freezer: Speeds the precipitation reaction with low precipitation reaction with low amounts of DNAamounts of DNA

DNA…DNA…the code of lifethe code of life

What do we know about What do we know about DNA?DNA?• Structure:Structure:

Composed of nucleotides Composed of nucleotides (monomer) consisting of:(monomer) consisting of:

1) phosphate group1) phosphate group

2) deoxyribose sugar2) deoxyribose sugar

3) one of four nitrogen 3) one of four nitrogen basesbases

What do we know about What do we know about DNA?DNA?

• Structure:Structure:

Nitrogen bases are Nitrogen bases are named:named:

- adenine (A)- adenine (A)

- guanine (G)- guanine (G)

- thymine (T)- thymine (T)

- cytosine (C)- cytosine (C)



• The structure of these The structure of these nucleotides determines nucleotides determines how they fit together. how they fit together.

• Adenine fits with Adenine fits with ThymineThymine

• Guanine fits with Guanine fits with CytosineCytosine

What do we know about What do we know about DNA?DNA?• Structure:Structure:

• DNA is “double-DNA is “double-stranded”stranded”

• The nucleotides are The nucleotides are linked together linked together covalentlycovalently

• Phosphate – Sugar – Phosphate – Sugar – Phosphate – Sugar etc.Phosphate – Sugar etc.

• This is the “backbone”This is the “backbone”



• The two strands are The two strands are oriented in opposite oriented in opposite directionsdirections

• The two strands are The two strands are wound around each wound around each other forming the other forming the “helix” structure“helix” structure


• Function:Function:

• Codes for 80,000 Codes for 80,000 genes, which form genes, which form proteins…the building proteins…the building blocks of life.blocks of life.

EukaryoticEukaryotic DDeoxyriboeoxyribonnucleic ucleic AAcidcid

• DNA for ShortDNA for Short

• Double helixDouble helix - two strands made up - two strands made up of A, T, G, and C basesof A, T, G, and C bases

• Complex organisms - many Complex organisms - many linearlinear chromosomes (10,000,000,000 or chromosomes (10,000,000,000 or more base pairs)more base pairs)

Plant or Animal DNA Strand:Plant or Animal DNA Strand:

ProkaryoticProkaryotic DDeoxyriboeoxyribonnucleic ucleic AAcidcid

• Bacteria Bacteria - one - one circularcircular chromosome chromosome (1,000,000 base pairs) (1,000,000 base pairs)

• Chromosomes, in both cases, are held by Chromosomes, in both cases, are held by proteins to the cell or nuclear membraneproteins to the cell or nuclear membrane

• Most RNA is translated into proteins that Most RNA is translated into proteins that have have structural or functional jobsstructural or functional jobs in in cellscells

Bacterial DNA StrandBacterial DNA Strand

Let’s get our samples now and Let’s get our samples now and continue on with our continue on with our isolation…isolation…• Centrifuge: Spins solution at high Centrifuge: Spins solution at high

speed to concentrate DNA at the speed to concentrate DNA at the bottombottom

• TE: buffer at pH 8.0 TE: buffer at pH 8.0

• RNAse: enzyme which removes RNA RNAse: enzyme which removes RNA present in sample through digestionpresent in sample through digestion

Restriction Enzymes and Restriction Enzymes and GelsGels

Restriction EnzymesRestriction Enzymes

• Cut specific Cut specific sequences of DNAsequences of DNA

• Many different kindsMany different kinds

• Named after organism Named after organism they came from, they came from, enzyme numberenzyme number

• E.g. EcoR1E.g. EcoR1

Bacteria Produce Restriction Bacteria Produce Restriction EnzymesEnzymes

• Uniquely bacterial Uniquely bacterial protection protection mechanism…why?mechanism…why?

• Restriction enzymes Restriction enzymes are short are short nucleotidenucleotide sequences isolated sequences isolated from bacteria cells from bacteria cells that protect them that protect them from virus.from virus.

http://library.thinkquest.org/C0118084/Glossary/nucleotide.htm

Bacteria Produce Restriction Bacteria Produce Restriction EnzymesEnzymes• When a viral DNA enters When a viral DNA enters

the bacterial cell, the the bacterial cell, the restriction enzyme is restriction enzyme is able to recognize a able to recognize a specific sequence specific sequence (restriction site) on the (restriction site) on the DNA molecule, which is DNA molecule, which is usually 4-8 nucleotides usually 4-8 nucleotides long. The restriction long. The restriction enzyme will cut the viral enzyme will cut the viral DNA at these sites and DNA at these sites and hence restrict the hence restrict the growth of the virus.growth of the virus.

Bacteria Produce Restriction Bacteria Produce Restriction EnzymesEnzymes

• Several hundreds of Several hundreds of these enzymes have these enzymes have been isolated from been isolated from various organisms and various organisms and most are available most are available commercially. These commercially. These enzymes are used to enzymes are used to cut a segment of gene cut a segment of gene from a human DNA from a human DNA molecule.molecule.

DNA FingerprintingDNA Fingerprinting


• DNA fragments are DNA fragments are separated using gel separated using gel electrophoresiselectrophoresis

• Each band Each band represents the DNA represents the DNA which has been cut which has been cut into smaller pieces into smaller pieces using restriction using restriction enzymesenzymes

Gel ElectrophoresisGel Electrophoresis

From your studies of DNA, can From your studies of DNA, can you tell me what charge DNA you tell me what charge DNA

has?has?


• Gel is made of Gel is made of water and agarose water and agarose

• Wells on one end Wells on one end are where gels will are where gels will be loaded with our be loaded with our samplessamples

Gel ElectrophoresisGel Electrophoresis• The gel box contains The gel box contains

water and buffer to water and buffer to keep the pH constantkeep the pH constant

• Gel box has platinum Gel box has platinum wire that conducts wire that conducts protons and electronsprotons and electrons

• Gel box will be wired to Gel box will be wired to the power source the power source following the loadfollowing the load


• To the strand of DNA To the strand of DNA moving through the moving through the agarose, the gel agarose, the gel looks like a big looks like a big mesh-like mazemesh-like maze

• The DNA travels The DNA travels through the maze as through the maze as fast as it’s size will fast as it’s size will allowallow


• DNA moves from DNA moves from the negative the negative towards the positivetowards the positive

• Smaller – fasterSmaller – faster• Larger – slowerLarger – slower

• Where will these Where will these three end?three end?

Gel ElectrophoresisGel Electrophoresis• Review:Review:

** DNA travels – to ** DNA travels – to ++** When the power ** When the power

supply turns supply turns off, off, we can see we can see where where the bands the bands are and are and infer infer which are which are bigger and smallerbigger and smaller** Small goes far** Small goes far** Large goes not far** Large goes not far

DNA Fingerprinting DNA Fingerprinting Questions and AnswersQuestions and Answers

Do you know the answers to Do you know the answers to these questions?these questions?


• How good (accurate) is it at How good (accurate) is it at identification. For example, is it identification. For example, is it as good as classical fingerprints?as good as classical fingerprints?

Question 1:Question 1:

• How good (accurate) is it at How good (accurate) is it at identification. For example, is it as identification. For example, is it as good as classical fingerprints?good as classical fingerprints?

• Answer: In theory, with the exception Answer: In theory, with the exception of identical twins, of identical twins, EVERYONEEVERYONE on this on this planet has a different DNA fingerprint. planet has a different DNA fingerprint. That is, DNA fingerprinting That is, DNA fingerprinting ISIS as good as good (distinctive/unique/specific) as classical (distinctive/unique/specific) as classical fingerprinting for identification. fingerprinting for identification.

Question 2:Question 2:• What are its advantages?What are its advantages?

• Answer: In theory DNA fingerprinting will work with Answer: In theory DNA fingerprinting will work with much smaller amounts of material than a classical much smaller amounts of material than a classical fingerprint & DNA lasts much longer than classical fingerprint & DNA lasts much longer than classical fingerprints. DNA-containing samples that are many fingerprints. DNA-containing samples that are many years old (up to 25 million yr.) are still usable. Only years old (up to 25 million yr.) are still usable. Only very tiny quantities of DNA are required in order to very tiny quantities of DNA are required in order to carry out a highly accurate test. For example, dried carry out a highly accurate test. For example, dried blood, semen, spit, skin etc. on samples stored in blood, semen, spit, skin etc. on samples stored in dusty files for years are still usable. Samples of dusty files for years are still usable. Samples of mixed DNA's can also be used. DNA containing mixed DNA's can also be used. DNA containing evidence is much harder to clean up at a crime evidence is much harder to clean up at a crime scene than other evidence, like classical fingerprints. scene than other evidence, like classical fingerprints.

Question 3:Question 3:•What are its limitations?What are its limitations?

• Answer: There currently are no accepted Federal Answer: There currently are no accepted Federal standards for controlling the quality of DNA testing standards for controlling the quality of DNA testing nationwide. Poor quality & poorly controlled testing nationwide. Poor quality & poorly controlled testing can lead to can lead to QUESTIONABLEQUESTIONABLE and and SHODDY SHODDY RESULTSRESULTS. .

• Even if there is a perfect match between DNA, you Even if there is a perfect match between DNA, you can not say can not say HOWHOW the DNA containing sample got the DNA containing sample got there or there or WHENWHEN. In the O.J. trial a . In the O.J. trial a VALIDVALID question question was raised about the possibility of evidence being was raised about the possibility of evidence being planted. What makes this charge so powerful is the planted. What makes this charge so powerful is the EXTREME SENSITIVITYEXTREME SENSITIVITY of the procedure. of the procedure.

Now, how do we come up Now, how do we come up with those different with those different

bands?bands?

Answer: Restriction EnzymesAnswer: Restriction Enzymes

Let’s do an example of Let’s do an example of DNA Fingerprinting DNA Fingerprinting

together…together…

DNA Fingerprinting DNA Fingerprinting Example:Example:

• Two men fitting the description of a Two men fitting the description of a robber were caught in the vicinity of robber were caught in the vicinity of the crime. Both had cuts on their the crime. Both had cuts on their arms which they “explained away.” arms which they “explained away.” DNA samples were taken from each DNA samples were taken from each suspect and from the broken window suspect and from the broken window at the scene of the crime. at the scene of the crime.

DNA Fingerprinting DNA Fingerprinting Example:Example:• Using DNA Fingerprinting and Using DNA Fingerprinting and

Restriction Enzymes, we can Restriction Enzymes, we can determine which of the men was the determine which of the men was the robber!!robber!!

• We can cut each sample (one from We can cut each sample (one from each suspect and one from the crime each suspect and one from the crime scene) with two different enzymes, scene) with two different enzymes, run them on a gel and compare the run them on a gel and compare the resultsresults

So, how do we organize what So, how do we organize what we know? We organize the gel we know? We organize the gel lanes…lanes… LaneLane Description:Description:

11 DNA sample from DNA sample from crime scenecrime scene cut w/ Enzyme 1 cut w/ Enzyme 1

22 DNA sample from DNA sample from crime scenecrime scene cut w/ Enzyme 2 cut w/ Enzyme 2

33 DNA sample from DNA sample from Suspect 1Suspect 1 cut with Enzyme 1 cut with Enzyme 1




Fingerprinting Gel Projected Fingerprinting Gel Projected Results:Results:

Practical applications of Practical applications of DNA technologyDNA technology

Practical applications of DNA Practical applications of DNA technologytechnology

• Diagnosis of Diagnosis of diseases includes:diseases includes:– Huntington’s Huntington’s – PKUPKU– cystic fibrosis cystic fibrosis – Duchenne’s muscular Duchenne’s muscular

dystrophy dystrophy


• Human gene Human gene therapy therapy

• Somatic cell therapy Somatic cell therapy versus germ cell versus germ cell therapy therapy


• Pharmaceutical Pharmaceutical products:products:

InsulinInsulin

human growth human growth hormonehormone

• Protection from viral Protection from viral infection infection


Forensic usesForensic uses

DNA fingerprintingDNA fingerprinting

RFLPs and simple RFLPs and simple tandem repeats tandem repeats (microsatellite DNA (microsatellite DNA repeats of different repeats of different lengths) lengths)


• Environmental uses:Environmental uses:

• Genetically Genetically engineered microbes engineered microbes for mining, cleaning for mining, cleaning up toxic wastes, etc. up toxic wastes, etc.


Agricultural usesAgricultural uses• Animal husbandry Animal husbandry

– Transgenic animals Transgenic animals – Gene knock-in or Gene knock-in or

knock-out animals knock-out animals (requires homologous (requires homologous recombination) recombination)

– Cloned animals Cloned animals

• Genetic engineering Genetic engineering in plants in plants – Can grow many plants Can grow many plants

from a single cell from a single cell

Working with DNA: Isolation and Fingerprinting. Funding and support received from…

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