DNA Fingerprinting Pawson, PVMHS, 2015

Student learning objectives Explain how crime scene evidence is collected for DNA analysis. Demonstrate understanding of Chargaff’s rules. Explain why DNA separates the way it does on a gel. List 3 sources of DNA that could be collected from a suspect. Explain how DNA is amplified by using PCR. Explain what CODIS is and who uses it. Explain how DNA fingerprinting can be used to identify a parent, child, or other relative. Interpret the results of a gel.

Labs and activities Virtual DNA extraction lab Virtual PCR lab Virtual gel electrophoresis Blackett Family DNA activity (online) Exploratory CODIS lab

Vocabulary Pg. 159, 10 minutes Gene: Allele: Chromosome: DNA fingerprint: Short tandem repeat: Variable number tandem repeats: DNA probe: Restriction enzyme: PCR (polymerase chain reaction): Electrophoresis:

History of DNA fingerprinting DNA has been used since the 1980s to investigate crimes, establish paternity, and identify victims of war or large-scale disasters. Most technologies for analyzing biological evidence, were originally developed for medical diagnostic or treatment purposes. DNA fingerprinting uses segments of DNA that are non-coding, otherwise known as “junk DNA.” These areas have a higher incidence of variability. DNA profiling is an important tool for forensics because no two people (except identical twins) have the exact same DNA.

Applications Solving crimes Identifying accident victims Identifying soldiers in war Paternity testing Inheritance claims Missing persons investigations Convicted felons databases – solving cold cases

Review of DNA Structure DNA is composed of nucleotides; which in turn are composed of a nitrogenous base, a phosphate, and a deoxyribose (a sugar). A DNA molecule is actually composed of 2 strands of DNA attached together by hydrogen bonds. Nucleotides match together in a specific manner: Adenine matches with Thymine Cytosine matches with Guanine

Chargaff’s rules Erwin Chargaff discovered that the % of guanine and cytosine were always the same in any given sample of DNA. Also the % of adenine and thymine were the same in any given sample of DNA. He concluded that these bases must always pair up as A-T and C-G.

Review of DNA Structure 1 molecule of DNA forms 1 chromosome. Humans have 46 chromosomes. A gene is a section on a chromosome that codes for a protein. An allele is a particular “version” of a gene. Often there are several alleles for any given gene, this is what allows for variation in populations. A molecule of DNA contains coding genes, but it also has areas that don’t code for anything at all. This is called “junk” DNA.

Epigenetics Sometimes there may be physiological variations that are not caused by differences in DNA… These changes occur due to environmental factors that affect the way genes may or may not be expressed. This is what accounts for differences between identical twins. The study of this phenomenon is called epigenetics.

Sources of DNA Any cell that has a nucleus contains DNA Blood – white blood cells Semen – sperm cells Saliva – white blood cells and cheek cells Hair – follicular tag Teeth – pulp Skin cells

Junk DNA The areas that do not code for proteins are sometimes called “junk” DNA. These areas often have a nucleotide sequence that just repeats itself over and over. These are called tandem repeats. Tandem: a group of 2 or more arranged one after another The length and number of repeats can be different from person to person. This is referred to as a polymorphism (many forms) and it differs from person to person.

Tandem repeats Variable number tandem repeat (VNTR): A nucleotide sequence of approximately 9 to 80 bases in length The number of repeats varies from person to person and is passed on just like a gene (remember this is “junk” DNA & doesn’t code for anything). Short tandem repeat (STR): A nucleotide sequence of approximately 2 to 18 bases in length These are much shorter than VNTRs The number of repeats varies from person to person and is passed on just like a gene. This is the most common type of “fingerprint” used for DNA profiling.

STRs AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote both alleles are the same length (7,7) or (8,8) Heterozygote alleles differ (7,8)

Position of Forensic STR Markers on Human Chromosomes CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA 13 CODIS Core STR Loci (FBI) D2S1338 D19S433 AMEL Sex-typing Penta E Penta D

Information on 13 CODIS STRs Locus Name Chromosomal Location Repeat Motif ISFH format GenBank Accession Allele in GenBank Allele Range Number of Alleles Seen * CSF1PO   5q33.3-34 TAGA X14720 12 6-16 15 FGA   4q28 CTTT M64982 21 15-51.2 69 TH01   11p15.5 TCAT D00269 9 3-14 20 TPOX   2p23-pter GAAT M68651 11 6-13 10 VWA   12p12-pter [TCTG][TCTA] M25858 18 10-24 28 D3S1358   3p [TCTG][TCTA] NT_005997 18 9-20 20 D5S818   5q21-31 AGAT G08446 11 7-16 10 D7S820   7q11.21-22 GATA G08616 12 6-15 22 D8S1179   8 [TCTA][TCTG] G08710 12 8-19 13 D13S317   13q22-31 TATC G09017 13 5-15 14 D16S539   16q24-qter GATA G07925 11 5-15 10 D18S51   18q21.3 AGAA L18333 13 7-27 43 D21S11 21q21 Complex [TCTA][TCTG] AP000433 29 24-38 70 * Butler, Forensic DNA Typing, Appendix 1

9 repeats of TCAT

Possible Genotypes in Offspring Inheritance of STRs Mother with 2 different STRs alleles per loci (9,12) Father with 2 different STR alleles per loci (14, 15) 9 12 15 14 Possible Genotypes in Offspring 9,14 9,15 12,14 12,15

Matching Tissue Matching Inheritance Matching Two samples that have the same band pattern are from the same person Inheritance Matching Each band in a child’s DNA fingerprint must be present in at least one parent

Electropherogram An electropherogram is a plot of results from an analysis done by electrophoresis. Allows for an easy way to view and compare results of DNA analysis & DNA profiles.

AMEL (amelogenin) Used to identify sex chromosomes Females (XX) have only one peak Males (XY) have two peaks STR within sex chromosome

DNA Databases CODIS: Combined DNA Index System Used primarily by the FBI, CODIS is a database of DNA profiles of any DNA ever collected in a criminal case. Ex. Rape kits This information is collected and placed into the database to be accessed by law professionals and available to cross reference with other cases. The data is submitted by labs across the US In addition to National Data (NDIS) CODIS may have international data There may also be private databases used for medical purposes, research studies, or population studies.

DNA Collection Use disposable gloves and collection instruments Avoid physical contact, talking, sneezing, and coughing in the evidence area Air-dry evidence and put it into new paper bags or envelopes Dry or freeze the evidence Keep evidence cool and dry during transportation and storage

Preparing DNA samples for fingerprinting Extraction: Before anything else, the DNA needs to be extracted from the cell nucleus. Cells are isolated, and the cell and nuclear membranes are digested. DNA can then be separated out from the other cellular components. Restriction: DNA must be cut into smaller segments. Special enzymes called restriction enzymes, cut DNA in specific places (like a pair of molecular scissors). Restriction enzymes recognize a unique pattern of DNA bases and cut the DNA at that location. Amplification: polymerase chain reaction (PCR) PCR amplifies (makes more of) the DNA that is used for fingerprinting. https://www.youtube.com/watch?v=0HCWmD7Mv8U

DNA Analysis Electrophoresis: the fragments of cut up DNA are then separated using gel electrophoresis. DNA fragments separate based on the size of the fragment. First, a gel is placed into a gel box & the DNA is loaded into the wells at the end of the gel closest to the negative charge. A standard (control, marker) is loaded into at least one well too. A power source is turned on sending an electric current through the box. DNA has a negative charge and will therefore move toward the positively charged end of the box. The DNA fragments will sort out based on size with the smallest fragments moving the easiest.

DNA probes DNA probes can be used to identify unique sequences in DNA. A probe is a synthetic sequence of DNA bases that are complementary to the sequence you are searching for. The probe can be “labeled” with a florescent dye that will glow that color when viewed under UV light. This allows the technician to focus on and compare the regions of interest only. Ex: DNA code is AAGCTTA, the probe will be TTCGAAT

VNTR vs. STR analysis VNTR uses restriction enzymes, whereas STR does not. STR DNA goes straight to PCR and the areas of interest are flanked by a primer and amplified. Each STR will be labeled with a different colored florescent dye. VNTR uses a traditional gel electrophoresis, STR uses capillary gel electrophoresis. DNA is still separated out by size, but it’s in a tube. As the STR passes the detector, the color is recorded. VNTR uses much larger pieces of DNA.

The ABI 310 Genetic Analyzer:

Fluorescent Emission Spectra for ABI Dyes 5-FAM JOE NED ROX ABI 310 Filter Set F 100 80 60 Normalized Fluorescent Intensity 40 20 520 540 560 580 600 620 640 WAVELENGTH (nm) Laser excitation (488, 514.5 nm)

Interpreting DNA analysis Bands are used to match samples of DNA DNA fingerprinting can match crime scene DNA with a suspect determine maternity, paternity, or match to another relative eliminate a suspect free a falsely imprisoned individual identify human remains

Any questions??