DNA Fingerprinting Please highlight all your answers with a yellow background or use a different color font that is easy to read LEARNING OBJECTIVES 1 Describe the structure and function of DNA 2 Describe the procedure of agarose gel electrophoresis and its application to DNA separation 3 Perform selective DNA digestion using restriction enzymes 4 Explain the importance of PCR in DNA sample preparation 5 Separate the DNA by electrophoresis and estimate the size of a DNA fragment 6 Evaluate and interpret various scenarios by which DNA fingerprinting is used as a biotechnology tool to identify individuals and show whether individuals or animals are related to each other INTRODUCTION This experiment introduces the basic concepts of DNA fingerprinting a method used in various medical and forensics procedures as well as in paternity determinations This protocol will demonstrate the similarities and differences in organisms at the genetic level The basic concept of any DNA fingerprinting protocol includes the extraction of DNA from any cell the restriction or cutting of the DNA by enzymes called endonucleases the amplification of the small amount of DNA collected and the analysis of the resulting DNA fragments using agarose gel electrophoresis Two basic techniques exist that allow analysis of DNA Polymerase Chain Reaction PCR is commonly used in molecular and genetic research Restriction Fragment Length Polymorphism RFLP analysis is another technique used to compare differences and similarities between individuals at the genetic level We will use the PCR technique in our investigation Many forensic scientists including those involved with criminal investigations use PCR analysis in order to eliminate potential suspects related to a crime scene For our experiment you will be given three DNA samples consisting of DNA collected from a crime scene and DNA collected from two suspects Your task will be to identify the true criminal using the gel electrophoresis method SCENARIO THE CRIME An extremely valuable molecular model that was made and used by famous scientists to figure out a key question in biology was going to be put up for auction It was expected to sell for a huge sum Two people offered early bids to purchase the model prior to the auction but the science institute that owns the model thought that the prices these people offered were too low In addition when news of the model s auction was announced a distant relative of one of the now deceased scientists came forward and laid claim to the model questioning the institute s right to sell it The night before the model was to be taken to the auction house the glass case housing the model was broken into and the model stolen It appeared that whoever broke into the case was cut on the sharp edge of the broken glass Although the thief tried to clean up the blood a very small amount was left on the edge of the glass There is footage from a security camera but the image quality is poor and the thief s face was covered The crime investigators began by questioning the two people who had tried to buy the model before the auction as well as the scientist s relative who had claimed ownership One of the two who made early offers had a solid alibi as to where she was during the time the model disappeared The scientist s relative also had a solid alibi However the second early bidder appeared very nervous when the authorities were at his home In addition his build is similar to that of the person seen in the security video and his hand has a large cut The authorities also found that one of the employees of the institute who also has a build like that of the person seen in the security video was working late and would have been present when the model was stolen He also has a significant cut on his hand but claims to have gotten it from a broken windowpane he was replacing in his home The investigators obtained a warrant and got blood samples from the two most likely suspects the late night employee and the nervous early bidder The investigators will compare the DNA profile of each of these suspects with the pattern obtained using the DNA isolated from the blood left on the glass case THE POLYMERASE CHAIN REACTION The polymerase chain reaction PCR is used to make many copies of a defined segment of a DNA molecule To perform PCR you first decide what DNA segment you wish to duplicate or amplify Then you obtain two short single stranded DNA molecules that are complementary to the very ends of the segment Each of the single stranded molecules must have a base sequence that is complementary to one specific location in only one strand of the target DNA and each one must be complementary to only one end of the segment These short single stranded molecules are the primers for PCR To begin the chain reaction a large number of primers is mixed with the target molecule in a test tube containing DNA polymerase enzyme buffer and many nucleotides The DNA polymerase used in PCR reactions is able to withstand very high temperatures the reason this characteristic is necessary to the reaction will become apparent later The buffer maintains the conditions under which the enzyme will work Nucleotides are the building blocks of DNA This mixture is heated to almost boiling so that the hydrogen bonds that hold the two strands of the parental DNA molecule together are disrupted and the two strands separate or denature Next the mixture is allowed to cool Ordinarily the two strands of the target DNA region would eventually line up and re form their base pairs However there are so many primers in the mixture that the short primers find their complementary sites on the target strands before the two target strands can line up correctly for base pairing Therefore a primer molecule base pairs hybridizes or anneals to each of the target strands Now DNA polymerase enzyme adds nucleotides to the 3 end of each primer using the bases on the target strand as a template New complementary strands are made with the 5 end of each being formed by a primer In this manner two double stranded DNA fragments are formed where before there was only one The cycle of denaturation hybridization and DNA synthesis is repeated many times Each time the number of DNA fragments in the mixture is doubled When this process is repeated 30 times a typical number of PCR cycles more than a billion copies of the DNA from the region between the two primers are generated PCR AND DNA FINGERPRINTING How is PCR