History of DNA Fingerprinting In 1984, Dr. Alec Jeffreys developed a technique for isolating and analyzing sequences of DNA He called this procedure DNA Fingerprinting In 1985, Dr. Kary Mullis invented the PCR technique allowing for the creation of a DNA profile from trace amounts of DNA

Human Genome The genome is the total amount of DNA in the nucleus of an organism Humans have about 3 million base pairs of DNA Most of the human genome is the same from one person to another, but there are variations The variations, which occur in the non-coding regions of the DNA, consist of unique patterns of end to end repeated base sequences called tandem repeats

Tandem Repeats The number and location of the tandem repeats are unique in each individual so they create a unique DNA profile These repeats may be studied to aid in the identification of individuals The more locations in an individual’s DNA that are examined, the higher the probability that you can identify the individual

Variable Number of Tandem Repeats (VNTRs) The number of copies of the same repeated base sequence in the DNA can vary among individuals Ex: the sequence ACTGACGATC might be repeated 3 times in one person, but 7 times in another person VNTRs can be 9 to 80 bases long

Short Tandem Repeat (STR) Short sequence of DNA, usually only two to five base pairs in length, within the non-coding DNA STRs are the preferred method of analysis because they are more accurate and can be used with small or partially degraded samples of DNA VNTRs are longer and require the DNA to be longer, making it difficult to separate the VNTR sequences

Uses for a DNA Profile Tissue Matching: used to match crime scene evidence to a suspect; the two samples must have the same band pattern Inheritance Matching: each band in a child’s DNA fingerprint must be present in at least one parent

DNA Directionality One of the parent strands in DNA runs in a 5’ to 3’ direction while the other runs in a 3’ to 5’direction The 3 and 5 refer to the carbon number of the deoxyribose ring

Primers and Polymerase DNA Primers are short segments of DNA that are complementary to the target DNA DNA Polymerase is the enzyme that binds free-floating nucleotides to the complementary bases on a DNA strand Restriction Enzymes are proteins that recognize a particular sequence in DNA and cut the DNA apart at that location

Steps in DNA Fingerprinting Extraction Restriction Fragments Amplification Electrophoresis

Extraction DNA must be removed from the nucleus of the cells

Restriction Fragments Restriction enzymes are used to cut apart the DNA at specific sites

Amplification Polymerase Chain Reaction (PCR) generates multiple identical copies from trace amounts of original DNA evidence Enable forensic scientists to make billions of DNA copies from small amounts of DNA in just a few hours

Steps in PCR Mix the primers with DNA, DNA Polymerase, buffer and nucleotides Heat the mixture to boiling to denature the DNA

Steps in PCR (cont.) Allow the mixture to cool At this point, the DNA would normally re-zip to form the original double-stranded molecule, but the primers attach to the DNA instead

Steps in PCR (cont.) DNA polymerase will now bind nucleotides to the end of each primer to complete the complementary strands There are now 2 complete copies of the DNA The entire process is repeated over and over again to create millions of fragments of DNA

Electrophoresis In this process, DNA fragments created through PCR are separated by using an electrical field DNA is negatively charged and will move towards a positive electrode The smaller the fragment, the faster it will travel

Steps in Electrophoresis Preparing the Buffer: add 25 ml of 20x TBE to 475 ml of distilled water Preparing the Gel: melt the agarose and let it cool Pour the Gel: place a comb into the gel box and pour the gel into the box so that it flows between the teeth of the comb; do not spill the gel into the areas at either end of the box; let the gel set

Steps in Electrophoresis Load the Gel: pour TBE solution into the gel box so that it covers the surface of the gel and floods the areas at the ends of the box; pull the comb out; using a pipette draw up the DNA and dye out of the tubes and load into the well

Steps in Electrophoresis Adding electrodes: use carbon filter paper as electrodes; use alligator clips to attach the electrodes to the power supply

Steps in Electrophoresis Running the Gel: turn the power on and let the gel “run”; do not disturb the box while the gel is running; once the blue dye reaches the end of the gel, turn off the power Staining the DNA: pour blue staining solution on top of the gel and let it sit 4 minutes; rinse the gel 3-4 times and leave the gel overnight to develop Destaining: destain the gel again; leave water in the box and change it about 4 times to gradually wash away the “background” stain

Southern Blotting The DNA on the gel can now be transferred to a nylon membrane in a procedure called Southern Blotting The bands of DNA on the membrane are the DNA fingerprint A radioactive piece of DNA called a probe is used to locate complementary sequences on the membrane

CODIS Combined DNA Index System: an electronic database of DNA profiles Individuals who have been convicted of certain crimes (i.e. rape, murder, child abuse) have their DNA profiles entered into the database