dna profiling is the use of molecular genetics methods to determine the exact genotype of a dna...

DNA profiling is the use of molecular genetics methods to determine the exact genotype of a DNA sample to distinguish one human being from another

Used in forensics to investigate crime scenes, missing persons, mass disasters, paternity, evolutionary links, …

Crime scenes often contain biological evidence (blood, semen, hair, saliva, bones, skin) from which DNA can be extracted

Humans have ~3 billion bp and >99.5% do not vary, however a small percentage (<0.5%) does differ

Varying regions are called “polymorphic” due to nt differences between one individual and another and polymorphisms are in regions of our chromosome (called loci) that do not have any known function - this DNA is satellite DNA (specifically short tandem repeats/microsatellite)

Crime Scene InvestigationDNA Profiling / DNA Fingerprinting

Ways to detect unique sequences within mammalian DNA

1. Restriction digestion of chromosomal DNA works for some organisms BUT not in mammals:EX: humans have 3 billion base pairs with 1 million restriction fragments formed from a single restriction enzyme digest, nt differences between people will result in different restriction digestion products - but TOO difficult to isolate a single band on a gel from this large number of fragments

2. To characterize a specific gene use blot hybridization


RFLPs

Genome of each of us is unique (exception: identical twins)Variation in sequences between individuals is most pronounced in DNA that does not code for proteinsHypervariable regions called “polymorphic sites”

Polymorphic sites - variation due to small insertions, deletions, or point mutations in restriction sites

SO .. Restriction fragment with a polymorphic site may differ from one person to the next resulting in:

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)Pattern of bands on a blot hybridization sometimes called a “DNA fingerprint” because can use it to identify an individual


History of Forensics

Early use of science for criminal investigations - use of photographs to document crime scenes

Past ~100 years - use of fingerprinting

First genetic evidence collected for investigations - blood group typing (A, B, AB, and O)Test is very fast and straight-forward. BUT 40% of population is type O, so not useful if several suspects are type O.

1980s - first use of DNA-based forensic test called restriction fragment length polymorphism analysis (RFLP); discovered by Alec Jeffries, focus on VNTRs (variable number of tandem repeats)


Restriction fragment length polymorphism analysis (RFLP)


Restriction fragment length polymorphism analysis (RFLP)

Although technique is powerful and has great discriminating potential, it is laborious, cannot be easily automated and is time-consuming

Depends on DNA sequences being several hundred base pairs in length, so DNA must be of reasonable quality (not degraded) and it requires a large amount of DNA (not always able to get enough DNA from the crime scene)

SO turn to using STRs - short tandem repeat DNA (smaller repeat lengths than VNTRs)Easier to analyze than RFLPs because:1. due to small size they are easily replicated by PCR2. Because they can be amplified by PCR, STR tests can be automated, with several tests being run

at the same time 3. Even degraded DNA can give OK results4. Even a single piece of hair (or a drop of blood, cheek cell, or piece of bone) can give enough

genetic material for successful analysis


Advances in PCR have made a huge impact

MODERN FORENSIC DNA PROFILING MAKES IT POSSIBLE TO DISTINGUISH ANY TWO PEOPLE ON THE PLANET (EXCEPT IDENTICAL TWINS), LIVING OR DEAD


Polymerase chain reaction (PCR)Denature dsDNAAnneal primers to now ssDNAPolymerization with thermostable DNA pol (DNA synthesis - get 2 copies from 1)

One cycle can be repeated over & overAfter cycle 30, > 1 billion identical molecules (230 = 1.07 x 109)


DNA profiling performed at many loci to improve the power of discrimination CODIS - COmbined DNA Index System - is a federally maintained database of DNA obtained from crime scenes and convicted violent offenders; started by FBI in 1998CODIS examines 13 loci, or markers, that are uniformly distributed across the human genomeAlso includes AMEL (determines gender)

The 13 loci reveal no medical or health info about a person; called “anonymous” markers


Why 13 loci?


This week’s LabScene: the Highway Motel, Room #13

The motel manager hears loud voices, a woman screams, and a shot rings out. The manager runs to the window in time to see the receding lights of a car leaving in a hurry. The door to room #13 is open. The manager runs to the open door to see a man lying face down in a pool of blood. He calls 911. The police arrive, and begin to examine the crime scene. An apparent homicide, but with no obvious clues as to who committed to crime. Or..? A forensic specialist is called in to examine the crime scene and evidence is collected. In addition, four suspects samples are collected.

You will perform PCR analysis on a single locus, the BXP007 locus - start this TODAY

Run PCR products on a 3% agarose gel, visualize products, compare to the allele ladder and to the crime scene sample, determine who committed the crime!

In addition to running your PCR products on the agarose gel you will also run your restriction digests from the genetic engineering lab.


Set up PCR in previous lab period:

Items needed:

Ice bath containing one yellow tube labeled MMP (blue liquid inside), 5 other tubes labeled CS (purple tube), A (green tube), B (blue tube), C (orange tube), and D (pink tube).

5 PCR tubes (these are smaller than we usually use, 0.5 microliter size)

5 normal size microfuge tubes used as adaptors (1.5 mL size)

Foam float

Marker

P20 pipettor

PCR block


Label PCR tubes (0.5 microliter size) “CS”, “A”, “B”, “C”, and “D”Also put your group name on the tube

Put 0.5 microliter tubes inside 1.5 mL tubes and put 1.5 mL tubes into foam float and set on ice, keep all samples on ice throughout the set up of these samples

Set up the tubes as follows:PCR tube label DNA template Master mix + primers* CS 20 microliters crime scene DNA (purple tube) 20 microliters MMP (yellow tube)A 20 microliters suspect A DNA (green tube) 20 microliters MMP (yellow tube)B 20 microliters suspect B DNA (blue tube) 20 microliters MMP (yellow tube)C 20 microliters suspect C DNA (orange tube) 20 microliters MMP (yellow tube)D 20 microliters suspect D DNA (pink tube) 20 microliters MMP (yellow tube)

Add all the DNA templates to their respective tubes first - change tips for each DNA!

Next add MMP to each tube, mix by flicking the tube and/or by gently pipetting the the solution up and down - use a fresh tip each time!!

I will start the PCR cycles up in my research lab

*MASTER MIX + PRIMERS contains Taq DNA polymerase buffer, MgCl2, dNTPs, Taq DNA polymerase, DNA primers specific for the BXP007 locus


PCR cycle:

STEP FUNCTION TEMP TIME # of CYCLESInitial denaturation Denature 94˚C 2 min 1 cycleThermal cycling Denature 94˚C 30 sec

Anneal 52˚C 30 sec Extend 72˚C 1 min

Final extension Extend 72˚C 10 min 1 cycle

Hold Hold 4˚C hours

} 35 cycles


On Wednesday - electrophorese PCR products

Materials needed:3% agarose gel with ethidium bromide5 PCR samples from before break1x TAE running bufferOrange G loading dye “LD” (orange liquid)Allele ladder “allele ladder” (also orange liquid!)P20 pipettorGel boxPower supply


Electrophorese PCR products1. Add 10 microliters of Orange G loading dye “LD” to each PCR reaction tube (5 of these); mix well

2. Set up gel apparatus, rotate gel from pouring position to loading position, add 1x TAE to gel box, carefully pull out comb

3. Load 20 microliters of each sample into wells on the gel

Lane 1 Allele ladder 20 uLLane 2 crime scene 20 uLLane 3 suspect A 20 uLLane 4 suspect B 20 uL Lane 5 suspect C 20 uLLane 6 suspect D 20 uL

4. Run gel at 120 volts for ~45-60 minutes (do not let orange dye front migrate off the gel)

5. Visualize results on UV light box


Study questions:

1. Why do you need to perform PCR on DNA evidence from a crime scene?

2. Did your samples all generate PCR products? If not, give reasons to explain why.

3. Does the crime scene DNA sample have a genotype that matches any of the suspects? If so, which one matches?

4. If you had a pool of 13 suspects and only one suspect had a genotype that matched the BXP007 locus found at the crime scene would you be satisfied that you had identified the perpetrator? Why or hy not? Explain.

USEFUL WEBSITES

Folding of RNA moleculeshttp://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi

Molecular Biology toolshttp://www.molbiol.net/http://www.molbiol.net/biolinks/alphabar/PCR%2520and%2520Primer%2520Design.shtmlhttp://ntdb.chem.cuhk.edu.hk/tools.htm

Oligonucleotide calculatorhttp://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.htmlhttp://www-medlib.med.utah.edu/masspec/mongo.htmhttp://www.schepartzlab.yale.edu/

Protein informationhttp://www.basic.nwu.edu/

Chemistry calculatorshttp://users.pandora.be/educypedia/education/calculators.htm

Good websites of “tools”http://www.csb.yale.edu/people/steitz/Toolkit/toolkit.htmhttp://szewczak.com/

Plasmid informationMacPlasmap

http://www-medlib.med.utah.edu/masspec/mongo.htm

dna profiling is the use of molecular genetics methods to determine the exact genotype of a dna...

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