DNA FINGERPRINTING MADE EASY FOR FORENSICS

Transcription

1 DNA FINGERPRINTING MADE EASY FOR FORENSICS Presented by Eilene Lyons The St. Louis Community College Florissant Valley Biotechnology Program Some slides are from a downloaded PPT presentation from The National Institute of Standards and Technology (NIST) 1

2 Where do we get the DNA? Blood Semen Saliva Urine Hair Teeth Bone Tissue 2

3 DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Individual nucleotides 3

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5 REVIEW OF DNA STRUCTURE NUCLEOTIDE monomers A, T, G, C DNA POLYMER - long chain of nucleotides HYRDROGEN BONDS hold two polymers together to form a ladder COMPLEMENTARY nucleotides make the steps of the ladder A bonds with T G bonds with C 5

6 Review of Replication Two polymers separate Each serves as a template DNA polymerase adds nucleotides, but not without a primer 6

7 DNA: Nucleotide structure A nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar (either ribose or 2'-deoxyribose), and one to three phosphate groups. Together, the nucleobase and sugar comprise a nucleoside. The phosphate groups form bonds with either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site most common. Cyclic nucleotides form when the phosphate group is bound to two of the sugar's hydroxyl groups.[1] Ribonucleotides are nucleotides where the sugar is ribose, and deoxyribonucleotides contain the sugar deoxyribose. Nucleotides can contain either a purine or pyrimidine base. Nucleic acids are polymeric macromolecules made from nucleotide monomers. In DNA, the purine bases are adenine and guanine, while the pyrimidines are thymine and cytosine. RNA uses uracil in place of thymine. Ribose structure indicating numbering of carbon atoms 7

8 DNA Polymerase requires a primer WHY? 8

9 DNA Polymerase requires the primer s 3 OH to add the next nucleotide 9

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11 Review of DNA Replication POLYMERS SEPARATE - Hydrogen bonds break to unzip the sides of the ladder TEMPLATE one polymer of the DNA PRIMERS complementary RNA added to the template NUCLEOTIDES the monomers (A, T, G, C) DNA POLYMERASE the enzyme that adds new complementary nucleotides starting at the 3 OH end of the RNA primer 11

12 Assessment 1. Name four molecules required to make new DNA. Template DNA Primers Nucleotides (A, T, G, C) DNA Polymerase 13

13 Polymerase Chain Reaction: DNA Replication in a test tube STEP 1: DENATURATION HEATING TO 94 C STEP 2: PRIMER ANNEALING COOLING TO 58 C primer primer STEP 3: EXTENSION HEATING TO 72 C primer primer FIGURE 3. Steps of PCR 14

14 DNA Amplification with the Polymerase Chain Reaction (PCR) Starting DNA Template Separate strands (denature) Forward primer 5 3 Add primers (anneal) Make copies (extend primers) Reverse primer 15

15 PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created 16

16 Assessment 2. Nucleotides PCR primers polymers complementary ladder steps DNA polymerase template 17

17 STRs are Polymorphisms Small Tandem Repeats Polymorphism: two or more distinct forms of a section of DNA AATT 7 repeats 8 repeats 18

18 People are Diploid We inherit polymorphisms from our parents. Everyone has 2 copies of each piece of DNA: One chromosome from Mom One chromosome from Dad 19

19 TPOX The STRs used for DNA Fingerprinting D3S1358 D5S818 FGA CSF1PO D8S1179 D7S820 TH01 VWA AMEL D13S317 D16S539 D18S51 D21S11 AMEL 20

20 STRs can be replicated in a test tube. chromosome cell nucleus Double stranded DNA molecule Target Region for Replication Individual nucleotides 21

21 PCR Replicates STRs Polymerase Chain Reaction What must be added to the tube? 1. Template DNA 2. Primers 3. DNA Polymerase (Taq from Thermus aquaticus) 4. Nucleotides 22

22 Forensics labs use Multiplex PCR many STRs copied at once 23

23 DNA has a negative charge. If put into an electric field, DNA will migrate towards the positive electrode. ROPE TRICK 24

24 Viewing the DNA Stain with ethidium bromide fluoresces in UV light 26

25 Analysis of a Single DNA Polymorphism PCR results of amplification of the D1S80 Range of fragment sizes = 224 to 640 bp 400 bp 27

26 Looking at many polymorphisms at once 28

27 Check out these sites: html ex.htm 29

28 DNA replication Diagram (From Wikipedia, not part of EL presentation) 30