Introduction to DNA DNA Fingerprinting and the Polymerase Chain Reaction

1. Introduction to DNA DNA Fingerprinting and the Polymerase Chain Reaction

2. The Cell • Smallest unit of life • Compose all living things • The “nucleus” (one of many organelles) contains genetic information the cell needs to exist and reproduce • - most cells organize genetic information into chromosomes

3. Chromosomes • our body’s way of organizing all the information that our genetic material contains • 23 pairs in humans • - each pair contains one from mother and one from father

4. Genes • Each chromosome contains 100s to 1000s of information blocks called genes • Each gene is the blueprint for a specific protein in the body • - may tell our body what color our eyes are supposed to be, dozens of proteins are responsible for synthesis of ATP, digesting food, etc, etc etc

5. DNA • Each chromosome and every gene is made of deoxyribonucleic acid (DNA) • DNA is a polymer of repeating units called nucleotides • Each nucleotide contains three parts • - phosphate group • - sugar (deoxyribose) • - nitrogenous base

6. Nucleotides Phosphate Sugar The nitrogenous base is always one of four molecules: adenine guanine cytosine thymine

7. The DNA Backbone • Nucleotides are linked together with alternatingP-S-P-S-P……..

8. The DNA Double Helix • DNA is normally double stranded • The two nucleotide chains are held together by hydrogen bonds • A always pairs with a T on the other strand; C always pairs with G

9. The DNA Double Helix The two strands wrap around each other to form helical structure shown (double helix)

10. Functions of DNA • Two primary functions • - transmit information from one generation to the next • - provide blueprint for making proteins the same way every time Two Types of DNA • nuclear or chromosomal DNA (inherited from mother and father) • mitochondrial DNA (inherited from mother only)

11. DNA Replication • DNA is unwound • An enzyme called DNA Polymerase adds complementary bases to “single stranded” • - A with T • - C with G

12. Restriction Enzymes Enzyme that cuts DNA at specific sequences Recognize and binds to 6-8 nucleotide stretch

13. Gel Electrophoresis • Gel with different sized pores • agarose and acrylamide are common materials • Load DNA samples into wells at top of gel • Run electric current through the gel • DNA moves due to negative charge • Smaller bands run “faster”

14. Sequence Repeats in the Human Genome • Repeat polymorphisms (satellites) are short segments of DNA that repeat a few to thousands of times and are found at specific locations in human DNA • There are many types of repeat polymorphisms that occur on human chromosomes • Each individual will have different numbers of these repeats at each of these spots – the numbers of repeats at each location in you are a random combination of the repeats in your parents • Each of these spots, or loci, are given different names. The most common are: • variable number of tandem repeats (VNTR) • amplified fragment length polymorphism (AmpFLP) • short tandem repeats (STR) • single nucleotide polymorphism (SNP)

15. Sequence Repeats in the Human Genome Variable Number of Tandem Repeats (VNTR): repeats of 9 to 80 base pairs (bp), total length is 500 to 23,000 bp, very specific due to length and repeats, testing is expensive and time-consuming, degrade in older DNA samples due to random breaking of DNA strands Amplified Fragment Length Polymorphisms (AmpFLP): repeats of 8 to 16 bp, total length 100 to 1300 bp, shorter and less susceptible to degradation, first loci to be used in forensic analysis Short Tandem Repeats repeats of 2 to 7 bases, total length 100 to 400 bp, shorter yet thereby less susceptible to breakage, these loci are the current standard in forensic laboratory analysis, ideal size for PCR amplification Single Nucleotide Polymorphisms (SNP): a single base change as a result of mutation, not commonly useful to forensic investigators, can be potentiallyused to distinguish identical twins

16. Polymerase Chain Reaction (PCR) • Technique devised in 1983 to amplify small amounts of DNA • Can be performed on DNA from a single cell • - cigarette butt, a licked stamp, root of a single hair, 1/50,000 a drop of blood (0.1 microliters) • The amplified DNA can then be used to: • - identify a suspect or victim • -determine an individual’s sex • -determine species (if not human)

17. PCR to Amplify a Person’s DNA • Steps Involved: • Isolate repeating loci from a person’s DNA using restriction enzymes • Design primers – short segments of synthetic DNA that are complementary to DNA on either side of the VNTR regions

18. Add vast excess of the primers and heat mixture to 75 oC • This causes DNA strands to separate by breaking hydrogen bonds between bases

19. 4. Cool to 15 oC. Primers hydrogen bond (anneal) to complementary strands 5. Add DNA polymerase and all four types of nucleotides. The polymerase (enzyme used in DNA replication) will fill in the rest of the two strands.

20. You now have two identical copies of the DNA you started with. 6. Repeat steps. Heat to break hydrogen bonds. Cool to anneal more primers (still there in vast excess). Allow DNA polymerase to fill in the remaining strands. Two strands of DNA become four. Etc…Etc…Etc…..

21. PCR Originally, the DNA polymerase would have to be added between each heating step because it would fall apart at 75 degrees. Now, an enzyme called Taq DNA polymerase is added. This is a very stable enzyme isolated from bacteria living at thermal vents in the ocean (up to 95 oC) In just 32 rounds of PCR, 1 copy of DNA becomes 4.2 billion copies. This would take about 3 hours to perform in lab. PCR Animation

22. DNA Fingerprinting Used to identify individuals by their repeat regions (usually STR) regions: Steps involved: • Isolate and amplify DNA if needed • DNA is cleaved into smaller pieces with restriction enzymes • DNA is separated with gel electrophoresis

23. DNA is transferred to a nylon membrane (Southern blotting) • A radioactive primer is designed that will be complementary to unique regions (STR, etc, regions). Add this to nylon membrane containing DNA. • Wash off excess primer and hold nylon up to a photographic plate to expose. The pattern will be unique to the individual.

24. Clearly, suspect one is the match….. If all STR regions are considered, there is a one in 3.4 billion chance of error. This means there may be one other person on the planet that would be too similar to tell the difference. If all other satellite regions are also considered, the chances of error go way, way down… 1 in 53,581,500,000,000,000,000

25. Mitochondrial DNA • genetic material from the mitochondria (cellular organelle where energy is produced) • inherited from the mother only • Advantages: • more sensitive (less DNA needed), degrades slower than nuclear DNA • can be used in cases where nuclear DNA cannot (hair without root, skeletal remains) • Disadvantages: • all people of same maternal line will be indistinguishable (less discriminatory) • more work, more time consuming, more costly

26. CODIS – Combined DNA Index System • National software developed by the FBI • Distributed to local, state, and national crime labs • All 50 states mandate inclusion of DNA fingerprint (if available) from violent and sexually motivated crimes • Mostly a database of STR regions • Thousands of matches have led to the capture of criminals that otherwise would not have been caught • This has led numerous people to suggest a national DNA database that would include only polymorphism information…