DNA Profiling,(DNA fingerprinting),DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus,Model of DNA DNA is Comprised of Four Base Pairs,What is DNA Profiling?,A technique used by scientists to distinguish between individuals using only samples of their DNA,Who Invented it?,The process of DNA fingerprinting was invented by Alec Jeffreys at the University of Leicester in 1985.,Stages of DNA Profiling,Stage 1: Cells are broken down to release DNA If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR),Stages of DNA Profiling,Stage 2: The DNA is cut into fragments using restriction enzymes. Each restriction enzyme cuts DNA at a specific base sequence called the restriction site.,DNA Restriction Enzymes Discovery,Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Endonucleases (restriction enzymes) cleave DNA strands In simpler terms, bacteria use them to cut up the viral DNA Over 3,000 known enzymes,Enzyme Site Recognition, Each enzyme digests (cuts) DNA at a specific sequence called a restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (read the same both backward and forward) (eg GAATTC),Palindrome,Restriction site,Fragment 1,Fragment 2,Common Restriction Enzymes,EcoRI Eschericha coli 5 prime overhang,Pstl Providencia stuartii 3 prime overhang,An EcoR1 restriction enzyme,Restriction enzymes,Restriction endonucleases http:/highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf:535:535:/sites/dl/free/0072437316/120078/bio37.swf:Restriction%20Endonucleases cleave DNA at specific points,Stages of DNA Profiling,The sections of DNA that are cut out are called restriction fragments. This yields thousands of restriction fragments of all different sizes A tandem repeat is a short sequence of DNA that is repeated at a specific chromosomal locus (location) These repeats are very unique to each individual,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).,Stages of DNA Profiling,Stage 3 Agarose gel electrophoresis. Electrical current carries negatively-charged DNA through gel towards positive (red) electrode,Power Supply,Buffer,Dyes,Agarose gel,Agarose Gel electrophoresis,DNA is placed at one end of a gel An electrical current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones,Stages of DNA Profiling,DNA is separated on basis of size. Notice the shorter fragments make it through the gel quicker than the longer fragments,Stages of DNA Profiling,A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. A photographic copy of the DNA bands is obtained.,Stages of DNA Profiling,Stage 4: The pattern of fragment distribution is then analyzed.,Analysis of Stained Gel,Determining restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples,Molecular Weight Determination,Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0,Fingerprinting Standard Curve: Semi-log,Uses of DNA Profiling,DNA profiling is used to solve crimes and medical problems,Crime,The DNA profile of each individual is highly specific. The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).,Biological materials used for DNA profiling,Blood Hair Saliva Semen Body tissue cells DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse.,DNA Profiling can solve crimes,The pattern of the DNA profile is then compared with those of the victim and the suspect. If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (it does not prove they committed the crime). If the profile doesnt match the suspect then that suspect may be eliminated from the enquiry.,Example,A violent murder occurred. The forensics team retrieved a blood sample from the crime scene. They prepared DNA profiles of the blood sample, the victim and a suspect as follows:,Was the suspect at the crime scene?,Suspects Profile,Blood sample from crime scene,Victims profile,Famous Cases,Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.,Did suspect 1, suspect 2, or the boyfriend leave the sperm collected as evidence? Do the female cells found at the crime scene belong to the victim? Note that the more markers you use to cut the DNA, the more bands you will get. This increases the ability to tell different people apart.,Famous