History of DNA Fingerprinting

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. Steps in DNA Fingerprinting • Extraction • Restriction Fragments • Amplification • Electrophoresis

11. Extraction • DNA must be removed from the nucleus of the cells

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

13. 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

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

15. 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

16. 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

17. 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

18. 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

19. 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

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

21. 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

22. 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

23. 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