2Table of Contents Introduction Pretest Part 1 – Historical Information Part 2 – Electrophoresis Equipment SetupPart 3 – Preparing DNA Sample for ElectrophoresisPart 4 – Set up and run a DNA GelConclusion – Additional introductory resourcesPosttest – Administered by instructor in class
3IntroductionThis is an introductory tutorial on DNA Fingerprinting and ElectrophoresisDuring this tutorial, you will examine:Historical timeline of DNA manipulationComponents of DNA manipulationElements of electrophoresis
4Pretest Prior to continuing the tutorial, complete the pretest. Click on the “pretest” link below which will bring up a Word document.Print out the pre-test, complete the pretest and return it to your instructor.Then, proceed with the tutorial on electrophoresis.PRETEST
5Part 1 - What is a gene?Each chromosome is an extremely long DNA molecule that can be thousands or millions of base pairs long.The vast length of chromosomes posed a problem for scientists who were trying to isolate and study the stretches of DNA that make up genes.
6Part 1 – Molecular Scissors – Pg 1 In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for the existence of "molecular scissors" that could cut DNA. He showed that E. coli bacteria have enzymes that recognizes and destroys foreign DNA.Arber and his associates called this group of DNA-cutting enzymes restriction endonucleases (sometimes referred to as restriction enzymes). They showed that restriction endonucleases are specific in their snipping: a given enzyme will cut DNA only when it recognizes exactly the right sequence.
8Part 1 – Molecular Scissors – Pg 3 Restriction Enzyme = BamHIRecognition Site = G G A T C CCuts = Notice the cut occurs at the arrows and follows the red lineRestriction Enzyme = HaeIIIRecognition Site = G G C CCuts = Notice the cut occurs at the arrows and follows the red line
9Part 1 – Molecular GlueNot long after Arber's discoveries, Arthur Kornberg identified the pasting mechanism for DNA, an enzyme he called ligase.Kornberg was trying to construct artificial viral DNA from viral fragments, but had been unable to make a biologically active molecule. Once he added ligase, however, he found that the enzyme made it possible to paste the ends of DNA molecules together.
10Part 1 – Cutting and Gluing DNA MolecularScissorsRestrictionEnzymeMolecularGlue
11Part 1 – Checkup Chromosomes consist of: Long strand of DNA.The molecular scissors which cut DNA into small pieces are:Restriction endonucleases.Where do these molecular scissors cut the DNA?At a specific sequence of bases.For example, what is the sequence where EcoR1 cuts?G A A T T CThe molecular glue that will bind pieces of DNA together.LigaseClick or space bar to continue with next unit
12Part 2 – Electrophoresis Equipment What is Gel Electrophoresis? Electro = flow of electricity, phoresis, from the Greek = to carry acrossA gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatinGel electrophoresis refers to the separation of charged particles located in a gel when an electric current is appliedCharged particles can include DNA, amino acids, peptides, etc
13Part 2 – Electrophoresis Equipment Why “do” a gel electrophoresis? When DNA is cut by restriction enzymes, the result is a mix of pieces of DNA of different lengthsIt is useful to be able to separate the pieces - i.e. for recovering particular pieces of DNA, for forensic work or for sequencing
14Part 2 – Electrophoresis Equipment What is needed to do this? Agarose - a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of cross linked moleculesAgarose is the material used to make the gel we will use.
15Part 2 – Electrophoresis Equipment What is needed to do this? – Pg 2 Buffer - in this case TBEThe buffer provides ions in solution to ensure electrical conductivity.Not only is the agarose dissolved in buffer, but the gel slab is submerged (submarine gel) in buffer after hardening
16Part 2 – Electrophoresis Equipment What is needed to do this? – Pg 3 Also needed are a power supply and a gel chamberGel chambers come in a variety of models, from commercial through home-made, and a variety of sizes
17Part 2 – Electrophoresis Equipment How does it work? DNA is an organic acid, and is negatively charged (remember, DNA for Negative)When the DNA is exposed to an electrical field, the particles migrate toward the positive electrodeSmaller pieces of DNA can travel faster in a given time than larger pieces because smaller fragments do not experience as much resistance when traveling through the gel.
19Part 2 – Electrophoresis Equipment Apparatus – Another Example Positive electrodeCombAgarose gel blockDNA loaded inwells in the agaroseBufferBlack backgroundTo make loading wells easier
20Click or hit the space bar to continue to the next page Part 2 – Checkup – Page 1A suspension, gelatin like, used to separate DNA fragmentsgelName of the material used to make our gels.agaroseLiquid containing ions which promote electrical conductivitybufferName the apparatus containing the gel slab and buffer.gel chamberWhat charge does DNA possess?negativeDNA will migrate toward which terminal in a gel chamber?positiveClick or hit the space bar to continue to the next page
21Click or hit the space bar to continue to the next page Part 2 – Checkup – Page 2Why do large fragments move slower?because they are biggerClick or hit the space bar to continue to the next page
22Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 1 DNA can be extracted from almost any human tissue.Buccal cells from inside cheek for paternity tests.Sources of DNA at crime scene: blood, semen, hair follicle, saliva.DNA extracted from evidence is compared to DNA from known individuals
23Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 2 Extracted DNA molecules are combined with restriction enzymes (endonucleases) and allowed to react with each other.Restriction enzymes are produced by bacteria as a defense against viruses.These enzymes cut DNA at specific base sequences called recognition sites.Results in smaller pieces of DNA called RFLP’s.
24An EcoR1 restriction enzyme Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 3An EcoR1 restriction enzyme
25Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 4 RFLP Analysis:RF stands for Restriction Fragments. Those are the fragments that were cut by restriction enzymes.L stands for Length, and refers to the length of the restriction fragment.P stands for Polymorphisms, a Greek term for “many shapes”. The lengths of some of the restriction fragments differ greatly between individuals.RFLP = Restriction Fragment Length Polymorphism
26Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 5 Electrophoresis of these RFLP’s produce different patterns of DNA bands.With 3 billion base pairs in the human genome, however, RFLP analysis would produce a “smear” of many similar sized fragments.smear
27Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 6 Molecular biologists have identified regions of the human genome where restriction fragment lengths are highly variable between individuals.
28Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 7 VNTR alleles are highly variable regions of human DNA.VNTR stands for ‘variable number of tandem repeats.A tandem repeat is a short sequence of DNA that is repeated at a specific chromosomal locus.Tandem repeats are interspersed throughout the human genome.The number of repeats at a given place on a certain chromosome is highly variable from one person to another.The number of such repeats is usually different on the paternal and maternal members of the same person’s chromosome pair.
29Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 8 Red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. (Here 3 different variants, may be 50 in reality).
30Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 9 Analysis of a VNTR locus most commonly results in a two-band pattern, one band inherited from each parent.A one-band pattern can occur if the size of the two parental bands are the same or nearly the same.For our simple example of three different alleles designated A, B, and C illustrated above, six unique DNA profiles are possible.
31Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 10 The possible genotypes are AA, BB, CC, AB, BC, and AC
32Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 11 The RFLP markers most commonly used for DNA profile analysis are found on chromosomes 1, 2, 4, 5, 10 and 17.These RFLP markers are named after their locations on these chromosomes.For example, the marker on chromosome 2 is called D2S44 (section 44 of chromosome 2).These chromosomal locations are also referred to as DNA loci (plural) or locus (singular).
33Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 12
34Part 3 – Preparing DNA Sample for Electrophoresis – DNA Extraction – Page 13 DNA profiles vary from person to person.When profiles form a single VNTR locus from unrelated individuals are compared, the profiles are normally different.However, it is possible for two individuals to have the same profile at one or two loci.But the chance of more than one person having the same DNA profile at 4, 5, or 6 different VNTR loci is extremely small.
35Part 3 – Checkup – Page 1 What human cell(s) can we extract DNA? From almost any cell.What is used to cut the DNA into fragments?Restriction enzymes.How does a restriction enzyme know where to cut the DNA?Looks for specific sequence of bases, called recognition sitesThe resulting small pieces of DNA are called?RFLP’sWhat is the recognition site (sequence of bases) for EcoR1?G A A T T CWhat does RFLP stand for?Restriction Fragment Length PolymorphismClick or hit the space bar to continue to the next page of checkup questions
36Part 3 – Checkup – Page 2When you electrophores these fragments, what results?Different patterns of DNA bandsIf you electrophores all human DNA, what results?A smear of indistinguishable bands.What have scientists found that seems more unique among individuals?Regions of human DNA fragments that are highly variableThese highly variable areas are called what?VNTR’sWhat does VNTR stand for?Variable number of tandem repeatsCan you expect the VNTR’s at a location to be different from one person to another?Yes, the repeats at a given place on a certain chromosome is highly variable from on person to the next.Click or hit the space bar to continue to the next page of checkup questions
37Click or hit the space bar to continue to the next page Part 3 – Checkup – Page 3What chromosomes are usually tested for VNTR’s?Chromosomes 1, 2, 4, 5, 10 and 17.When analyzing DNA from different individuals, why use several chromosomes?More samples used from an individual, the less chance there will be duplication.Click or hit the space bar to continue to the next page
38Part 4 – Set up and run a DNA Gel Page 1 DNA is prepared by digestion with restriction enzymesAgarose is made to an appropriate thickness (the higher the % agarose, the slower the big fragments run) and “melted” in the microwaveThe gel chamber is set up, the ‘comb’ is insertedThe agarose may have a DNA ‘dye’ added (or it may be stained later). The agarose is poured onto the gel block and cooled
39Part 4 – Set up and run a DNA Gel Page 2 The comb is removed, leaving little “wells”.Buffer is poured over the gel, covering it completely.The DNA samples are loaded into the wells using a micropipette
40Part 4 – Set up and run a DNA Gel Page 3 Gel box top is secured.Terminal cords are attached to appropriate terminals.The power source is turned on and the gel is run.
41Part 4 – CheckupWhat happens when the percent of agarose used in the gel is changed?High the percentage, slower the fragments will move.Why is a comb used when preparing the gel?Makes the wells in the gelWhat is the micropipette used for?Load DNA samples into the wellsClick here to return to the contents pageOr click anywhere else or hit space bar to go to the next page
42ConclusionVideo Clip – Sam Sheppard Case – Application of DNA fingerprinting (Viewing video instruction: when web page appears: (1) click on “view” button, (2) On “required registration” screen, click on “Take a Test Drive” button, (3) Click on “view” button again.) Note: If you do not have sound, click on the “captions on” option. This will show the text as the video plays.)Use of DNA in Identification – Link to more detailed explanation of using DNA when comparing evidence DNA with suspect DNA.How can RFLP’s help find faulty genes? – Link to a description of another practical use of RFLPsDNA Fingerprinting, Genetics and Crime: DNA Testing and the Courtroom – A seminar by geneticist and evolutionary biologist Julian Adams of the University of Michigan explains the principles, procedures and issues involved with the use of DNA as a tool in the identification of individuals.