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Honors Forensic Science Chapter 4 – “DNA Fingerprinting” Mr. Davis
Name: ______________________________________________ Date: 10/26/17 Section: ___
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Chapter 4: DNA Fingerprinting, Study Guides, Projects, Research of Biotechnology
The concept of DNA fingerprinting, Steps of DNA Fingerprinting , DNA Gel Electrophoresis .
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1 Broughton High School
Name: ______________________________________________ Date: 10/26/17 Section: ___
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Chapter 4 – Vocabulary Words – DNA Fingerprinting
Vocabulary for Chapter 4
Term Definition
- Allele
- Chromosome
- DNA Fingerprint
- DNA Probe
- Electrophoresis
- Gene
7. PCR
- Restriction Enzyme
- STR
10. VNTR
- Polymorphisms
- DNA Analysis
- Annealing
- Denaturing
15. DNA
Polymerase
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The concept of DNA fingerprinting.
Only 0.001% of DNA (about 3 million bases) differs from one person to the next. However, those small variable regions are enough for scientists to generate a DNA profile of an individual, using DNA extracted from blood, bone, hair, and other body tissues or products.
In criminal cases, DNA is extracted from both the crime scene evidence and from the suspect. Both sets of DNA are analyzed for the presence of a set of specific DNA regions (markers). Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in the sample. A series of radioactive probes bound to a DNA sample creates a distinctive pattern for an individual.
Forensic scientists compare these DNA profiles to determine whether the suspect’s sample matches the evidence sample. A single marker is not usually unique to an individual, so forensic scientists generally look at multiple markers. If the sample profiles don’t match, the person did not contribute the DNA at the crime scene, but if the two DNA samples match at multiple regions, the odds are good that the two samples come from the same person. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim, especially if there are multiple probes. Four to six probes are recommended.
Pose the following question: “How small do the odds have to be when conviction of the guilty or acquittal of the innocent lies in the balance?” Tell students that many judges consider this a matter for a jury to take into consideration along with other evidence in the case. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are only about 50:50. The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing.
This is a realistic depiction of a sexual assault case with gel electrophoresis. It could be seen that suspect 1's DNA band matches with the sperm DNA evidence. This means that the results are conclusive: the sexual offender is suspect 1 as suspect 2 and the boyfriend do not have DNA that matches with the collected Evidence. However, in different cases when there are no matches, suspects are able to be eliminated. In some cases, the results may be inconclusive as the process may not be successful or not enough DNA was collected.
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DNA Fingerprinting
The process of DNA fingerprinting is used to identify certain people and to also find out if people are related. The process includes the extraction of DNA as the first step. Stains and tissue is collected in order to extract DNA such as blood and saliva through buccal swabs (cheek). Depending on the type of stain or the tissue that is collected, the method of extraction and purification would vary. Epithelial tissues are known to be one of the easiest types of tissue of deal with while bone tissue may be slightly more difficult. DNA extraction with semen mixed in with other components such as vaginal epithelial cells (usually in sexual assault cases) has a particular method in extraction as the process must not mix the substances or cells together. This process is known as differential extraction. Differential extraction is based on the knowledge that sperm cells have protein disulphide bonds around them, making them resistant to enzymes that break up cell membranes that are usually used in DNA extraction for epithelial tissues. To break the bonds, an agent called Diatheothreitol is used since it is a "de-protecting" or disulphide bond reducing substance for thiolated DNA. In this process, Diatheothreitol is added to the DNA of the sperm and they are separated by filtration or Chromatography, depending on the type of Diatheothreitol used (solid catalyst or liquid form). After collecting the DNA, the amount of DNA collected is then recorded as this is crucial because the amount of DNA collect can vary widely. To retrieve samples of STR that are in the DNA, the PCR or the Polymerase Chain Reaction method can exponentially amplify certain sections of DNA (in this case, STR) so that the scientist can have thousands to millions of copies of the STR that are the same. The PCR method is based upon the method of thermal cycling, which is the process of heating and cooling down in a cycle. The PCR method consists of around 20-30 cycles. Short synthetic pieces of DNA called primers flank the DNA and indicate the sections of DNA that would be copied during the process. When starting, if the equipment was used and not cleaned up properly, it is heated until all the DNA and the primers have melted. The first stage is called melting or denaturing. The DNA will then be heated at a certain temperature so that the hydrogen bonds that connect the strand together would break and the DNA would separate into two and the primers would have been separated. The next step is called the annealing step which is when the temperature is cooled down to about so that the primers can attach to each of the DNA strands. The last step is called elongation or synthesizing. The DNA-Polymerase then fills in the missing strands by starting at the primer. The temperature at this stage depends on the DNA-Polymerase itself as well as the length of the DNA that is going to be amplified. The cycle is then repeated numerous times with new primers replaced and fresh Polymerase until many samples are replicated. The DNA-Polymerase could be added at the initial point of the process as well. This process is very effective in replicating copies of DNA. However, it should be ensured that the DNA is not replicated too much as this would affect the quality of the DNA strand as well.
The extracted and duplicated STR samples then go through a process called gel electrophoresis so that the DNA samples can be sorted and measured by length. The scientist slang for gel electrophoresis is "running the gel". This procedure features an agarose gel tray. The gel is made of hydrocolloid that is extracted from seaweed. At the top of the tray, there are small holes. The first step is to drop the DNA samples in each of the holes. Then, an positively charged electrical current is applied to the other end without the holes and negatively charged electrical current is applied to the end with the sample. As DNA is negatively charged because of its phosphate ions, the DNA would travel to the other end of the tray, which has a positive electrical charge. As shorter strands are less heavier than longer strands, they would travel faster and end up being closer to the end of the tray. DNA strands that have the same length and mass would end up being group together at the same area. As a single DNA strand cannot be seen by the naked eye, by using an electric current, DNA would end up being in groups that are called bands and therefore, can be seen. In order to increase the visibility of the bands, coloured dye or stains are added- preferably ethidium bromide, which is a chemical that can bind to DNA. The result can then be examined by the eye in order to find out various things such as whose father or mother is the child's or to link up DNA from the crime scene with a suspect. If the bands match each other, this means that the suspect was at the crime scene. In the circumstances of finding parents, the father and the mother's DNA strands would each match half of the child's DNA and so, if an alleged father has DNA that does not match the band of the child's, they are not the father.
Broughton High School DNA is found in the nucleus of cells in the human body. A perpetrator may leave biological evidence, such as saliva, blood, seminal fluid, skin, or hair, at a crime scene. This biological evidence contains the perpetrator’s unique DNA. Because this evidence is capable of identifying a specific person, it is known as individual evidence. A saliva sample can be collected from an envelope, a toothbrush, or a bite wound. DNA can be isolated from a sample of biological evidence as small as a drop of blood or a hair follicle.
When the amount of evidence left at a crime scene is very small, it is considered to be trace evidence. One of the problems encountered in dealing with trace evidence is that the evidence may be totally consumed during forensic testing. The use of the polymerase chain reaction (PCR) technique helps resolve this problem. Dr. Kary Mullis invented the PCR technique, for which he shared the Nobel Prize in 1993. PCR generates multiple identical copies from trace amounts of original DNA evidence. This enables forensic scientists to make billions of DNA copies from small amounts of DNA in just a few hours. The DNA produced with PCR can be analyzed using DNA fingerprinting techniques.
Steps of DNA Fingerprinting
Several steps are necessary before DNA samples can be analyzed and compared. These steps are summarized as follows and then expanded upon in more detail following the summary:
- Extraction. DNA is extracted from cells.
- Restriction fragments. In some VNTR analyses, DNA is cut by restriction enzymes. Restriction enzymes recognize a unique pattern of DNA bases (restriction sites) and will cut the DNA at that specific location. Restriction fragments of varying lengths are formed when the DNA is cut.
- Amplification. In the case of other VNTR analyses analysis, specifically chosen DNA fragments are amplified using polymerase chain reaction.
- Electrophoresis. DNA is loaded into the wells found in an agarose gel. When an electric current is passed through the gel, the negatively charged DNA fragments (pieces of DNA) migrate toward the positive end of the gel. DNA fragments are separated by size, with the smallest DNA fragments moving the fastest through the gel.
Extraction
The first step in preparing a sample for DNA fingerprinting is to extract the DNA from the cell nucleus. Cells are isolated from tissue and are then disrupted to release the DNA from the nuclear and cell membrane as well as from proteins and other cell components.
Restriction Fragments
In the case of some DNA profiles, after the DNA is extracted, the sample is mixed with a restriction enzyme to cut the long strands of DNA into smaller pieces called DNA restriction fragments. Restriction enzymes are “molecular scissors” that cut DNA at specific base sequences. Restriction enzymes are often produced and used by bacteria to defend themselves against invading viruses. There are many different restriction enzymes, and each one binds to a specific recognition site. Moreover, the enzyme cuts the DNA strand at specific locations within that restriction site. For example, the restriction enzyme Hind III recognizes the AACGTT base sequence. The Hind III restriction enzyme cuts the DNA between the two AA bases. When restriction enzymes cut DNA into pieces, fragments of many different lengths are produced. Within some of these fragments are unique sequences called VNTRs. Several different restriction enzymes may be used to cut the DNA in a sample.
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Amplification
With some DNA analyses, polymerase chain reaction (PCR) can be used to amplify certain pieces of the DNA that contain the VNTRs. In STR profiles, restriction enzymes are unnecessary and PCR allows the amplification of the strands with the STR sequences.
Electrophoresis
The fragments of cut DNA are separated by gel electrophoresis. A gel is the matrix (usually agarose) used to separate DNA molecules. Electrophoresis is the method of separating the molecules within an electric field based on the size of the DNA fragments. The gel forms a solid but porous matrix for the DNA fragments to move through. For this technique, the gel is placed into a gel electrophoresis chamber. Then, each DNA sample containing the amplified fragments is drawn up into a micropipette and placed into a separate well or chamber along the top of a gel. One well contains a control, a solution containing DNA fragments of known lengths called a DNA Ladder or Standard DNA. An electric current is passed through the gel. The negatively charged fragments of DNA in the wells move toward the positively charged opposite end of the gel. DNA fragments of different sizes are separated as the smaller DNA fragments move easily from the negative end of the gel toward the positive end of the agarose gel. All of the DNA fragments line up in bands along the length of the gel, with the shortest fragments forming bands closest to the positive end of the gel and the longest fragments forming bands closest to the negatively charged end.
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DNA Gel Electrophoresis
Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is
a common diagnostic procedure used in molecular biological labs.
Electrophoresis : The technique of electrophoresis is based on the fact that DNA is negatively charged at
neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it
will move toward the positive pole:
The rate at which the DNA will move toward the positive pole is slowed by making the DNA move
through an agarose gel. This is a buffer solution (which maintains the proper pH and salt concentration) with
0.75% to 2.0% agarose added. The agarose forms a porous lattice in the buffer solution and the DNA must slip
through the holes in the lattice in order to move toward the positive pole. This slows the molecule down. Larger
molecules will be slowed down more than smaller molecules, since the smaller molecules can fit through the
holes easier. As a result, a mixture of large and small fragments of DNA that has been run through an agarose
gel will be separated by size. This is a graphic representation of an agarose gel made by "running" DNA
molecular weight markers, an isolated plasmid, and the same plasmid after linearization with a restriction
enzyme:
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Worksheet No. 11
To Catch a Jewel Thief… DNA Fingerprinting
Purpose
To learn how to set up DNA digestions with restriction enzymes in order to create DNA fingerprints for analysis. To gain an understanding of restriction endonucleases critical for genetic engineering and biotechnology.
Importance (Application)
DNA Fingerprinting is a procedure whereby the genetic information, called DNA, in a person's cells is analyzed
and identified. The word fingerprinting is used because, just like a fingerprint, no two person's genetic code is
exactly the same. This makes DNA fingerprinting a very useful tool for our modern society.
It can be used to:
Determine Family Relationship - DNA can help find out who a person's parents or siblings are.
Prenatal paternity tests are available to mothers who need to identify the father of their unborn babies.
Detect Inherited Diseases - Your genetic code can be tested to determine your likelihood of getting
certain diseases.
Prove Guilt or Innocence - DNA left at the scene of a crime can be matched with a sample from a
suspect.
Identify a Dead Body - The DNA from an unidentified body can be matched with a person in a
government DNA database
Background
Introduction to DNA Gel Electrophoresis
In order to analysis DNA fragments restriction enzymes are used. Restriction enzymes are proteins that
cut long DNA pieces into smaller pieces or sections. Restriction enzymes cut DNA segments at particular
sections or specific sites in order to make the DNA fragments smaller. Restriction enzymes act like
microscopic scissors and cut DNA into segments. When DNA Electrophoresis labs are actually performed,
restriction enzymes are put into a small tube with DNA samples. The restriction enzymes digest the DNA into
smaller fragments. After the reaction is finished, it looks like a clear fluid.
In order for restriction enzymes digestion to mean much, you have to be able to somehow see the
different DNA fragments that are produced. So scientist separate DNA fragments or smaller pieces of DNA so
that they can look at the result of the restriction digestion by a process called Gel Electrophoresis. Gel
Electrophoresis takes advantage of the unique chemistry of DNA to be able to separate the DNA fragments.
Under normal conditions, the phosphate groups in the backbone of DNA are negatively charged.
According to the laws of electrical charges, opposites attract, so the negatively charged DNA molecules are
very much attracted to anything that is positively charged.
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To Catch a Jewel Thief: DNA Fingerprinting
Objective:
Read the passage below. Use the description to predict which suspect committed the crime: (Stealing the Golden Key)
The Set Up
The Key
On Friday October 6, 2017 the mayor of Raleigh, NC presented a large antique Golden Key to the City to the students at SMART Weekend for their outstanding academic achievement and citizenship. The Key was quite large and studded with semi-precious stones including amethyst and topaz. After a ceremony with Mr. Mitchell and the mayor awarding the key to the science scholars and SMART weekend students, the key was placed on a velvet cloth in a substantial display case just outside Broughton High School main office.
The Crime
Later that day, a great crash was heard from the hall outside Broughton High School main office. When several students went to investigate, all they found was an empty office and a broken display case. The GOLDEN Key to the City was gone!
The Evidence
The SMART Weekend students and the science scholars noticed that the intruder has cut him or herself on the broken glass of the display case. Here was a small puddle of blood left behind in the case. Being excellent science scholars, the students collected the blood for DNA analysis. The students knew how to get a DNA fingerprint of the suspect with the evidence. The students had performed a similar lab procedure with Mr. Davis just a week ago. The students took statements and collected DNA samples from each suspect. It worth noting that each suspect had a recent cut on his or her arm.
The Suspects
Suspect 1 - CIS President : The CIS President claims he was not in the office because he was returning from a late lunch. The cut on his right index finger was supposedly from a mishap serving birthday cake to a staff at lunch today.
Suspect 2 – Senior Director : The Senior Director was the only person alone in the office at the time of the break-in. She had a bright red stain on her wrist. She told the students it was from assisting Mr. Davis in a science experiment earlier in the day.
Suspect 3 – Director : The Director was delivering books to classrooms located down the hall from the main office. Therefore, she was in the general area at the time of the crime. It is a well-known fact that she passionately collects jewelry containing semi-precious stones. The Director had a Band-Aid on her right hand.
Suspect 4 - Lead Teacher : The lead teacher was wearing a jacket although it was a very warm day. The jacket appeared to conceal a bandage. It was possible the antique key was of interest to their collection of artifacts. The lead teacher’s coffee break was about the time the key was stolen. The lead teacher had ample opportunity to conduct the heist.
Thinking Critically
Who stole the Golden Key?
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CASE STUDIES
Ian Simms (1988)
Helen McCourt was last seen alive as she boarded a bus on her way home from work in Liverpool, England. Evidence found in the apartment of Ian Simms, a local pub owner, linked him to McCourt’s disappearance. His apartment was covered with blood, and part of McCourt’s earring was found there. The rest of her earring was found in the trunk of his car. Bloody clothing belonging to McCourt was found on the banks of a nearby river. Her body was never recovered. Dr. Alec Jeffreys analyzed the blood found in Simms’s apartment and matched it to blood from her parents. Dr. Jeffreys determined that there was a high probability that the blood found in Simms’s apartment matched that of Helen McCourt. Simms was found guilty of murder, and sentenced to life imprisonment. This was the first time DNA evidence was used to convict a murderer in a case where the victim’s body was not found.
Kirk Bloodsworth (1984)
Dawn Hamilton, age nine, was found raped and beaten to death in a wooded area near her home in 1984. In 1985, Kirk Bloodsworth was accused and convicted of the crime, despite evidence supporting his alibi. Because of a legal technicality, his case was retried, and he was again found guilty in 1986. He was sentenced to three terms of lifetime imprisonment. Bloodsworth continued to maintain his innocence. In 1992, a semen sample from the victim’s clothing was analyzed by both a private laboratory and the FBI laboratory. Using PCR and DNA fingerprinting, both laboratories determined that Bloodworth’s DNA did not match the DNA evidence from the crime scene. He was pardoned after spending nine years in prison.
Think Critically
Review the Case Studies and the information on DNA Fingerprinting. Should DNA evidence alone be sufficient to convict when there is no corroborative evidence? State your opinion and provide support for it.
Crime-Scene Investigation – Case 1
Crime-Scene Investigation – Case 2
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Mini Lab No. 2 - DNA Gel Electrophoresis
Kool-Aid Gel Electrophoresis
Introduction
In this lab, gel electrophoresis will be used to separate the dyes present in different flavors of Kool-Aid. In this experiment students will be introduced to the basis of gel electrophoresis. Students will learn about the basis of color theory and how the eye perceives color.
- Students will use dyes from Kool-Aid that will be exposed to an electrical current and a buffer solution.
- The Kool-Aid and the buffer solution will become ionized and take on a structure that will respond to an electrical charge. This lab contains eight different flavors of Kool-Aid: grape, black cherry, cherry, strawberry, tropical punch, lemon-lime, orange, and lemon.
Materials:
Each group of 6 students will perform the experiment. The materials needed to perform this lab include:
Disposal cups Agars gel solution Set of Kool-Aid samples with 50% glycerol 5 syringes, 1cc 5 yellow tips Electrophoresis tray with 1% agarose gel solution Set of (5) 9Volt batteries Set of Alligator clips – (one red cable & one black cable) Paper towels Gel Electrophoresis Diagram
Procedure:
- Collect the necessary equipment from the lab table
- Remove the plastic wrap from the 1% agarose gel
- Remove a 1cc syringe from the color plastic cup
- Carefully place a yellow pipette tip on the syringe, make sure the small plastic tube surrounding the syringe tips is not pushed to far up on the syringe
- Place 50 ml of water into the colored container
- Practice removing about 0.05 mls (1 drop) of water into and out of the syringe
- Use the 1-cc syringe and a yellow tip to load about 10μL into separate wells on the gel. Load each Kool- Aid sample only once. The first line on the yellow pipet tip is 10μL. TIPS
Steady syringe over well using two hands Be careful to expel any air in the pipet tip end before loading the gel. (If air bubbles form “cap” over well, sample will flow into buffer around edges of well.) Dip pipet tip through surface of buffer, position it over well, and slowly expel the mixture. Glycerol in the Kool- Aid solution weighs down the sample, causing it to sink to the bottom of the well. Be careful not to punch tip of the pipet through bottom of gel.
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DNA Gel Electrophoresis Technique
The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed.
Agarose gels are easily cast and handled compared to other matrices, because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator
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