DNA Profiling/Fingerprinting PDF

DNA PROFILING OR DNA FINGERPRINTING:  DNA PROFILING OR DNA FINGERPRINTING: DNA PROFILING (DNA FINGERPRINTING) describes the process by which individuals can be identified and compared based on their DNA sequence or DNA profile. This process requires matching an unknown sample of DNA...

DNA PROFILING OR DNA FINGERPRINTING:  DNA PROFILING OR DNA FINGERPRINTING: DNA PROFILING (DNA FINGERPRINTING) describes the process by which individuals can be identified and compared based on their DNA sequence or DNA profile. This process requires matching an unknown sample of DNA with a known sample to see if they correspond (make a match). When DNA fragments are cut with restriction endonucleases, fragments of different lengths can be resolved using gel electrophoresis and create the DNA Fingerprint  DNA PROFILING OR DNA FINGERPRINTING: If, after separation by gel electrophoresis, the pattern of bands formed by two samples of DNA fragments are identical, it means that both sources came from the same individual. If patterns are similar, it means that the two samples are most probably from two related people. At a crime scene, DNA is everywhere. It is present in all kinds of evidence collected at the scene, including blood, hair, skin, saliva and semen. Scientists can analyze the DNA in evidence samples to see if it matches a suspect's DNA.  HOW CAN DNA PROFILING BE USED TO IDENTIFY AN INDIVIDUAL? WHY DOES DNA PROFILING/ FINGERPRINTING WORK? RECALL: The chemical structure of everyone's DNA is the same. All humans are all made from the same DNA nucleotides, and contain the same amino acids and proteins. Almost every cell in our bodies contains DNA, the genetic material that programs how cells work **FYI - HOW CAN DNA PROFILING BE USED TO IDENTIFY AN INDIVIDUAL? WHY DOES DNA PROFILING/ FINGERPRINTING WORK? 99.9 % of human DNA is the same in everyone, meaning that only 0.1% of our DNA is unique! Each human cell contains three billion DNA base pairs. Our unique DNA, 0.1% of 3 billion, amounts to 3 million base pairs. This is more than enough to provide profiles that accurately identify a person. The only exception is identical twins, who share 100 percent identical DNA. Within the human genome there are two regions found on the DNA: i) Coding regions called GENES (99.9%) that contain instructions for protein synthesis ii) Non-coding regions called SATELLITE DNA (0.01%) where its function is thought to be related to structural component of DNA SATELLITE DNA is the highly repetitive, short sequences of nucleotides (nitrogen bases) between the genes and have a different density from bulk DNA. Satellite DNA varies greatly between different individuals in the number and length of repeats called short tandem repeats (STRs) As individuals all have a different number of repeats in a given sequence of satellite DNA, they will all generate unique fragment profiles. These different profiles or banding patterns can be compared using gel electrophoresis Example: AATTCCG - DNA sequence may repeat several times. This is the STR's. Every individual may differ in the numbers of these repeating units. You may have 5 repeating STR's between two genes, whereas the person next to you may have 6 and someone else may have 33, it varies widely.  **FYI - WHY IS DNA EVIDENCE ACCEPTED IN A COURT OF LAW AS EVIDENCE? The only difference between people (or any animal) is the SATELLITE DNA which is short repeating sequences of bases. The number of repeats is unique to every individual. DNA fingerprinting works because individuals have naturally occurring variations (satellite DNA) in their DNA. NO two individuals (except identical twins) have the same DNA.  FYI - WHY IS DNA EVIDENCE ACCEPTED IN A COURT OF LAW AS EVIDENCE? DNA Profiling/DNA Fingerprinting method is used in identifying whether or not a sample of DNA comes from a specific person. Scientists analyze banding patterns of an individual’s DNA and match it to evidence found at a crime scene. DNA fingerprinting has had a tremendous impact on law enforcement.  FYI DNA is cleaved with restriction enzymes and cut into fragments of different lengths. DNA fragments are then separated by electrophoresis and compared to those from the crime scene. Since the introduction of this technology, experts agree that information obtained from DNA testing can identify an individual with an accuracy of 99.8%, which far exceeds other testing methods such as ABO blood typing. APPLICATIONS OF DNA PROFILING/ DNA FINGERPRINTING TECHNOLOGY: Paternity Cases Forensic or Criminal Investigations  Paternity Cases Comparing DNA of offspring against potential fathers. Another important use of DNA fingerprints in the court system is to establish paternity in custody and child support litigation. In these applications, DNA fingerprints bring an unprecedented, nearly perfect accuracy to the determination.  Paternity Cases Children inherit half of their alleles from each parent and thus should possess a combination of their parents’ alleles Who is the father? Male 1 or Male 2 Why do you know that Male 1 is the father?  Forensic or Criminal Investigations: Identifying suspects or victims based on crime- scene DNA. FBI and police labs in U.S. and Canada have begun to use DNA fingerprints to link suspects to biological evidence-blood or semen stains, hair, or items of clothing-found at the scene of a crime. since 1987, hundreds of cases have been decided with the assistance of DNA fingerprint evidence.  Forensic or Criminal Investigations: Suspect DNA should be a complete match with the sample taken from a crime scene if a conviction is to occur. Who was at the crime scene? Suspect 1 or suspect 2 How do you know that Suspect 1 is guilty? Example: Who was at the crime scene ? Example: Dino was at the crime scene! ADDITIONAL APPLICATIONS: Locate lost or missing children, Identification Identification of genetic disorders Developing Cures for inherited Diseases Personal Identification: Because every organ or tissue of an individual contains the same DNA fingerprint, the U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action. Diagnosis of inherited disorders: DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. Developing cures for inherited disorders: Research programs to locate inherited disorders on the chromosomes depend on the information contained in DNA fingerprints. By studying the DNA fingerprints of relatives who have a history of some particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question. This work is a necessary first step in designing an eventual genetic cure for these disorders. PROBLEMS with DNA Fingerprinting Technology: Although the possibility is extremely remote, two non-identical twin people could have the same genetic structure. It is possible that the technician could make a mistake (human error), or contamination. BENEFITS with DNA Fingerprinting technology: DNA evidence could help convict criminals who may escape prosecution because of a lack of evidence. In future, databanks will store DNA samples of all individuals for identification. Should DNA fingerprinting be used to convict criminals? DNA fingerprinting is NOT 100% accurate, but the evidence may be accepted by jurors because it is scientific. DNA evidence does not convict, it is just another form of evidence. Possible son Nope! If you are interested in criminology then check out some of these videos: Russian baby swap (1 min 45 sec) https://www.youtube.com/watch?v=9pGMBeztWjQ DNA technology in forensics (5 min) https://youtu.be/nPVkooi8m9I Forensic Files Season 2 episode 4 (21 min) https://www.youtube.com/watch?v=BRtgNOD0I_w 5th Estate David Milgaard(16 min video) https://www.youtube.com/watch?v=aILwMVg_GdY Another local case: Thomas Sophonow acquitted RECOMBINANT DNA: https://www.youtube.com/watch?v=8Dd7M9PGhgQ  RECOMBINANT DNA: In one of the most controversial areas of genetic engineering research, scientists are able to combine genes from unrelated species. This technology is called RECOMBINANT DNA, where genes can be extracted from the nucleus of one organism and spliced into the chromosomes of a new organism. The donated genes affect the traits of the new organism and form transgenic organisms.  RECOMBINANT DNA: o This technique involves the laboratory manipulation of different sources of DNA, by cutting or cleaving the DNA into fragments using restriction enzymes. o The fragments are then ligated together using DNA ligase. o Finally Recombinant DNA can be inserted into a host organism’s chromosomes using a gene gun and that organism will use the foreign DNA as if it was its own to produce desired gene products. STEPS in RECOMBINANT DNA 1ST STEP. USE RESTRICTION ENZYMES to CUT DNA: Isolate the foreign DNA containing the desired gene away from their surrounding genes. This is accomplished by using a restriction enzyme. STEPS in RECOMBINANT DNA 2nd STEP: DNA RECOMBINATION USING PLASMID VECTORS: Combine the desired gene from the DNA fragment with a piece of DNA from the recipient organism. DNA fragments cannot function on their own or do not readily become part of a host organism’s chromosomes. They must become part of the genetic material of a living organism before the genes can be activated. STEPS in RECOMBINANT DNA DNA fragments may be combined with bacterial DNA so that later it can be inserted into a bacterial cell Plasmids are small, circular pieces of DNA from a bacteria cell that can be used as VECTORS to carry segments of cellular DNA into host cells. Two biological vectors are bacteria and viruses. STEPS in RECOMBINANT DNA Plasmids are removed from the bacterial cell and cut with the same restriction enzyme used to produce the DNA fragments. These cuts on the plasmid and the DNA fragments will have matching “sticky ends” and can be joined together using ligase enzyme. Recombinant DNA has now been produced. STEPS in RECOMBINANT DNA STEP THREE: DNA INSERTION OR CLONING: Insert the recombinant DNA into the host organism and many copies or clones of the recombinant DNA are made. STEPS in RECOMBINANT DNA After the foreign DNA has been spliced into the plasmid, the plasmid is transferred by micropipette or using a gene gun and shot into a cell of the host organism. (Host organism can be bacterial cell, yeast cell, plant cell or animal cell) When the host cell prepares to divide, it copies the recombinant DNA along with its own DNA. The process of making extra copies of recombinant DNA is a form of cloning. CLONES are genetically identical copies. Recombinant DNA Video: https://www.youtube.com/watch?v=MIfDx417SDs& list=PLb0WW0k29aHpIR44YLwrQol_cgisaDw-B 4.5 minute video explaining recombinant DNA  CLONED ANIMALS: Cloning is the technique of producing identical copies of genes, cells or organisms. It is the production of genetically identical organisms from single cells. All members of a clone and their parents have the same DNA.  NATURAL CLONING: Asexual reproduction in animals or plants by fragmentation, binary fission, and budding Eg: starfish growing new body if it contains part of center disc, planaria fragmented their body parts to reproduce, identical twin babies.. etc. REPRODUCTIVE CLONING: Definition: A procedure in which a full living copy of an organism is made. It is the genetic duplication of an existing organism especially by transferring the nucleus of a somatic cell of the organism into an enucleated oocyte (egg) (surrogate mother is needed) Cloning is very useful if an organism has a desirable combination of characteristics and more organisms with the same characteristics are wanted.  THERAPEUTIC CLONING: Definition: A procedure in which damaged tissues or organs are repaired or replaced with genetically identical cells that originate from undifferentiated stem cells. Sometimes cloning is used to produce skin, or other tissues/organs used to treat a patient. Cloning Dogs (7 min 20 s) https://www.youtube.com/watch?v=DmHYUvmiXQI Eyes of Nye – Cloning! (24 min) http://www.teachertube.com/video/the-eyes-of-nye-clon ing-season-1-episode-4-412686  PLANT AND ANIMAL CLONING Most plants can be cloned quite easily from pieces of roots, stem or leaves. Animals cannot be cloned in the same way from parts of their bodies. If animal embryos are divided up at an early stage into several pieces, each piece can develop into a separate animal. (this happens naturally when identical twins are formed)  PLANT AND ANIMAL CLONING The first experiments involving cloning date back to 1950`s with experimentation involving frog embryos and tadpoles. Some 40 years later, in 1990`s, mice were cloned using nuclei of cells from mouse embryos. Video: Dolly the Sheep (13 min) https://www.youtube.com/watch?v=tELZEPcgKkE  ADULT MAMMAL REPRODUCTIVE CLONING The first successful reproductive cloning of an adult with known characteristics was in 1997 in Scotland, where the first cloned adult mammal was of a sheep called “Dolly”. This demonstrated that an adult mammal could be cloned using specialized cells rather than embryonic cells. Since the birth of Dolly, other teams of scientists have cloned a number of other animals using cells from adult donors. As scientists study how these cloned animals develop, evidence is mounting that a number of problems may be associated with animal cloning. Dolly for example showed signs of premature aging. Other cloned animals have shown problems with gene expression.  HUMAN CLONING: In 2001, a team of scientists at an American research facility announced the first successful cloned human cells. Researchers involved in human cloning distinguish between therapeutic cloning and reproductive cloning. Therapeutic cloning: is the culturing of human cells for use in treating medical disorders, and organ transplants  HUMAN CLONING: Reproductive cloning: is the development of a cloned human embryo for the purpose of creating a cloned human being. In all countries in the world, the cloning of human beings is illegal, this is due to ethical and religious reasons. The potential benefits of these processes must be weighed against legal, moral and ethical issues.  What are your personal thoughts on cloning? GENE THERAPY:  GENE THERAPY: Gene therapy is a medical procedure in which normal or modified genes are transferred into defective cells of an individual to correct genetic disorders. In theory, the transferred genes will allow the recipient`s cells to begin functioning normally by giving them the instructions for synthesizing the missing peptides, therefore reversing the symptoms of the genetic disorder. Drawback to gene therapy is that the gene does not stay active for long periods of time, or the cells themselves do not have long life spans, so the treatment must be repeated often. Although the field of gene therapy is still very new, extensive debate is already underway about some of the moral and ethical issues at stake. Gene therapy may someday make it possible to “correct” cells that contain defective genes and cure genetic diseases, or even allow parents to “design” children so that they carry certain genes and not others. Two Methods of Gene Therapy: 1. Gene Surgery: involves removing cells from individual and growing them in culture. These cells can be transformed with a correct gene and then injected back into the individual to help cure the disorder. 2. Gene Modification: Modifying viruses so that they cannot cause disease, and then attaching DNA containing a desired gene to the viral DNA. The patient is then infected with the viruses, which carry desired genes into cells and correct the genetic disorder. Human Genetic Engineering is the modification of an individual's genotype with the aim of choosing the phenotype of a newborn or changing the existing phenotype of a child or adult. It holds the promise of curing genetic diseases like cystic fibrosis and increasing the immunity of people to viruses. It is speculated that genetic engineering could be used to change physical appearance, metabolism, and even improve mental faculties like memory and intelligence, although for now these uses are relegated to science fiction, but could become a reality with “designer babies”. The use of embryonic stem cells could potentially be used for Growing skin to repair a serious burn Growing new heart muscle to repair an ailing heart Growing new kidney tissue to rebuild a Gene Therapy https://www.youtube.com/watch?v=bLI1Gfb0ynw (3 minutes)  VIDEOS: DNA Fingerprinting: https://www.youtube.com/watch?v=DbR9xMXuK7c Gene Therapy https://www.youtube.com/watch?v=bLI1Gfb0ynw https://www.youtube.com/watch?v=xKerTa8yLRM (decent video) Forensic Files Purebred Love https://www.youtube.com/watch?v=EfXFsxSytts Runaway Love https://www.youtube.com/watch?v=0t6Ehy5L0qA  Clustered Regularly CRISPR/Cas9 Interspaced Short Palindromic Repeats What have you heard about CRISPR already?  TedX - https://www.ted.com/talks/andrea_m_henle_ how_crispr_lets_you_edit_dna?language=e n#t-255660 **(5 minutes) - stop and write notes along the way  CRISPR/Cas9 If restriction enzymes are… then CRISPR/Cas9 is… Axes Laser Scalpel Scissors A pencil and eraser (and scissors) A delete button A find and replace function A list of possibilities An imagination full of possibilities How CRISPR lets you edit DNA: (5 mins) https://www.ted.com/talks/ andrea_m_henle_how_crispr_lets_you_edit_dna?language=en#t-311500  Get a recipe card from Mrs. J Answer the following on your card: What is something new that you learned about this process? What is the link to biotechnology unit? What is the link to the overriding theme of bioethics in this unit? You have 4 minutes starting... now!  Round Robin Get into groups of 4 or 5 Everyone must read out what they put down for each question, you have one minute per person NO cross-talk, no interrupting, let the person finish what they are saying You have 5 minutes... Go! As a group, decide on an answer to this question: What is your biggest takeaway?  Sharing Each group quickly explains their biggest takeaway from this lesson on CRISPR and that person stands up to do it The next group should stand up right when they are about to finish and start sharing their own Continue to the next group until all the groups are done  Read the notes on CRISPR technology (there are no blanks to fill in on the first couple pages) FYI This section will NOT be on your quiz but it is interesting to see where biotechnology is going in the future Utility and Limitations CRISPR-Cas9 has become popular in recent years. Researchers notes that the technology is easy to use and is about four times more efficient than the previous best genome-editing tool (called TALENS). In 2013, the first reports of using CRISPR-Cas9 to edit human cells in an experimental setting. Studies using in vitro (laboratory) and animal models of human disease have demonstrated that the technology can be effective in correcting genetic defects. Examples of such diseases include cystic fibrosis, cataracts and Fanconi anemia, according to a 2016 review article published in the journal Nature Biotechnology. These studies pave the way for therapeutic applications in humans. CRISPR technology has also been applied in the food and agricultural industries to engineer probiotic cultures and to vaccinate industrial cultures (for yogurt, for example) against viruses. It is also being used in crops to improve yield, drought tolerance and nutritional properties. One other potential application is to create gene drives. These are genetic systems, which increase the chances of a particular trait passing on from parent to offspring. Eventually, over the course of generations, the trait spreads through entire populations. Gene drives can aid in controlling the spread of diseases such as malaria by enhancing sterility among the disease vector (female Anopheles gambiae mosquitoes). Utility and Limitations In addition, gene drives could also be used to eradicate invasive species and reverse pesticide and herbicide resistance. However, CRISPR-Cas9 is not without its drawbacks. The many potential applications of CRISPR technology raise questions about the ethical merits and consequences of tampering with genomes. In the 2014 Science article, Oye and colleagues point to the potential ecological impact of using gene drives. An introduced trait could spread beyond the target population to other organisms through crossbreeding. Gene drives could also reduce the genetic diversity of the target population. Making genetic modifications to human embryos and reproductive cells such as sperm and eggs is known as germline editing. Since changes to these cells can be passed on to subsequent generations, using CRISPR technology to make germline edits has raised a number of ethical concerns. Utility and Limitations Other ethical concerns are more nuanced: · Should we make changes that could fundamentally affect future generations without having their consent? · What if the use of germline editing veers from being a therapeutic tool to an enhancement tool for various human characteristics? To address these concerns, the National Academies of Sciences, Engineering and Medicine put together a comprehensive report with guidelines and recommendations for genome editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 This is a new “miracle” gene editing technique It was discovered naturally occurring in bacteria as an immune system, but scientists discovered how to shift the function to gene editing It targets specific sections of DNA and cuts them It can be used to deactivate genes, fix mutations, cause mutations, or completely replace genes It is easy, cheap, accurate, and fast  Cas9 Protein This is the really impressive part of the CRISPR/Cas9 tool Even though it is very common to refer to this tool as CRISPR, the Clustered Repeats of DNA are only a part of the original natural system found in bacterial DNA. Scientists don’t even use that part of the system for their purposes. They definitely use Cas9 though. Cas9 just needs a piece of guide RNA bound to it, and it will seek out the exact spot of DNA and cut through both strands of DNA Think about these 3 questions while you read page 18 of the notes What are the limitations of the CRISPR/Cas9 tool? Will there be ethical dilemmas involved in its use? What is the difference between using CRISPR/Cas9 on some cells of a patient vs. using it on an embryo in IVF? I want you to highlight/underline one sentence from each paragraph that you think is the most important  Curing diseases caused by mutations Scientists have used CRISPR technology to cure a disease called type I tyrosinemia in mice. The disease is caused by a small mutation, and causes the livers of the effected mice to not be able to break down certain amino acids The cure involved injecting mice with a mixture of Cas9 with the correct guide RNA, and copies of the gene without the mutation This was just injected into the veins of the mice, it made its way to the liver and changed the DNA of some cells These newly “fixed” cells also replicate and replace some of the old cells Only 0.4% of liver cells were “fixed” by the CRISPR/Cas9, but after replicating, they accounted for about 1/3 of the liver, which was enough to restore the function of the liver If these mice reproduced after this procedure, would their offspring still be at risk for the disease? Why were the scientists able to inject into the veins of the mice instead of directly into the livers? Is this enough proof to start using this technology of human embryos?  Other Uses for CRISPR Research Increase crop yields Produce healthier foods – Have plants grow essential vitamins and nutrients Grow organs for injured humans Reduce pest populations and invasive species Can you think of other possibilities?  Co-inventor of the CRISPR/Cas9 tool How CRISPR lets us edit our DNA - Jennifer Doudna (15 mins) https://www.youtube.com/watch?v=TdBAH exVYzc&ab_channel=TED Look at your diagram in your notes  How far in the future are we from editing embryos with this technology? It’s already happened! A scientist in China edited a single gene in twin girls in 2018. SciShow: The First Gene-Edited Babies (6 mins) https://www.youtube.com/watch?v=1qx7x8X9wLw&app=desktop &ab_channel=SciShow Same story (3 mins) https://nationalpost.com/news/world/gene-edited-babies-china  Quiz: https://map-ed.org/maped/?q=13  CRISPR videos https://nationalpost.com/news/world/gene-ed ited-babies-china (3 min news report on Chinese scientist) https://www.youtube.com/watch?v=1qx7x8X 9wLw&app=desktop (7 min Hank Green) Take some time to form your own educated opinion: Advantages of Disadvantages of biotechnology: What biotechnology: What are are the benefits ? What the harmful effects? are the advantages? What are the alarming Any hopes for the dangers? Any concerns future? for the future? To Do…. Practice questions