DNA Criminalist and Court Appearance Margaret Aceves State of California Dept. Of Justice Jan Bashinski DNA Laboratory Today I would like to give you all a brief description of what DNA is, where it is found, and how it is used in forensic DNA typing.

If Case goes to Court, Our role as Criminalists is to present the facts to the jury Case approach is to leave sufficient evidence for re-analysis.

DNA Criminalist in court Deputy District Attorney qualifies DNA Analyst as an expert through their education, training and experience What is DNA is explained to the court and jury. The DNA Analysis is briefly explained Specific DNA results are presented Significances of match is explained

Qualifying as an Expert Education of a DNA Analyst A Bachelors degree in a Natural Science AND FBI requires specific courses to participate in CODIS Molecular Biology Genetics Biochemistry Training in statistics

Training of a DNA Analyst

Experience of a DNA Analyst How many cases completed How many times have you testified Results of Proficiency

DNA Basics What is DNA? DNA stands for Deoxyribonucleic Acid It is the genetic material found in all plants and animals. “Blueprint of Life” – It contains all of the information for passing genetic traits from one generation to the next generation. DNA stands for deoxyribonucleic Acid. It is the genetic material found in all plants and animals. DNA has been called the “blueprint of life.” This is because it contains all of the information needed for passing down genetic traits (like blue eyes or brown hair) from one generation to the next generation.

Where is DNA found in your body? Our bodies are made up of cells. The nucleus of each cell contains DNA. Where is DNA found in your body? Our bodies are made up of cells. DNA is found in the nucleus of each cell.

Types of Biological Evidence Blood Semen Saliva Hair Sweat Teeth Bone Tissue Urine Here are the different types of biological evidence that DNA can be obtained from. An important point I would like to make is that all the DNA in your body is the same no matter where it comes from. Important Point: All cells in your body have the same DNA.

How is DNA inherited? DNA is packaged in the form of chromosomes. Humans have 23 pairs of chromosomes. ½ is inherited from your Mother ½ is inherited from your Father So now I would like to talk about how DNA is passed down from one generation to the next generation. I just showed you that in the nucleus, DNA is packaged into chromosomes. We humans have 23 pairs of chromosomes in each our cells. Half of our DNA comes from our mother and half comes from our father.

Human Chromosomes Here is an actual picture (called a karyotype) of the 23 pairs of chromosomes. The chromosomes are numbered one through 22 and then there is X/Y pair of chromosomes that determines gender. Notice that chromosome number one is the largest and chromosome number 22 is the smallest. Also, notice that each chromosome comes as a pair. This goes back to what I said. ½ of the DNA comes from Mom and ½ comes from Dad. So one of the chromosome number ones came from Mom and the other chromosome number one came from Dad.

Terms to Understand Chromosome – Physical structure that DNA is packaged in. Humans have 23 pairs. Genetic Marker (locus) – specific location on a chromosome. Allele – Alternative possibility for a genetic marker. Before I go any further I would like to explain a few terms. We just covered chromosomes. On the chromosomes at specific locations are genetic markers. There are different types of genetic markers. Another word for genetic marker is locus. In the next couple of slides I’ll explain about the genetic markers we use in forensic DNA typing. The last item I will cover here is the word allele. An allele is an alternative possibility for a genetic marker or a gene. As an example of an allele, think about eye color. Some people have brown eyes, some have blue eyes and some have green eyes. These are all different possibilities for the gene that codes for eye color.

Forensic DNA Typing More than 99% of our DNA is the same (this is why we all have one head, two eyes, two arms, two legs, etc). Short tandem repeats (STRs) are used in forensic DNA typing. STRs vary from person to person by the number of the repeating sequences. Before I talk about the genetic markers used in forensic DNA typing I would like to point out that almost all of our DNA is the same from person to person. So in forensic DNA typing we are looking at areas of the DNA that we know vary from person to person. The genetic markers we use are called short tandem repeats. The short tandem repeats that we use are four building blocks long and they repeat over and over again. STRs are known to vary from one person to another by the number repeats. And that is what we are actually measuring – that difference in the number of repeats.

Short Tandem Repeats (STRs) AATG 7 repeats 8 repeats Here is an example of a short tandem repeat marker. Think of these two lines as one of the pairs of chromosomes that I showed you and the location we are looking at is one of the short tandem repeats markers that we are interested in. Remember that half of our DNA comes from our mother and half comes from our father. So the seven repeating sequence of DNA came from one parent and the eight repeating sequence from the other parent. At the end of our analysis this will be referred to as a 7,8 for this particular STR marker. the repeat region is variable between samples while the flanking regions are constant

Location of 13 CODIS Core STR Loci Sex-typing CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA Here is a diagram showing the chromosomes numbered one through 22 and the X and Y chromosomes. And here are the locations of the 13 STR markers that we test in forensic DNA typing. We also test for gender using the amelogenin marker. So now I would like to take a few minutes to describe how I actually go about analyzing DNA from biological evidence. AMEL Sex-typing

Process of Forensic DNA Analysis Document evidence Sample evidence Apply chemicals and heat to separate the DNA from the rest of the cellular components Purify DNA Determine concentration of DNA The first step in the process is to document the condition of the evidence. Usually the evidence is a stain of some sort like blood or semen. I cut a small piece of the stain and put it into a small plastic tube like the one shown here. Then I add some various reagents and apply some heat to breakdown the cell membranes and separate the DNA from the rest of the cellular components that I’m not interested in. Finally I purify the DNA. The next step is to find out what the concentration of the DNA is.

Actual Analysis Hours of on hands sample manipulation

How much DNA? qPCR Instrument to quantitate the DNA extract

Amplification of DNA Polymerase Chain Reaction (PCR) Areas of DNA are replicated millions of times. Once I have determined what the concentration of the DNA is I need to make lots of copies of the 13 STR markers. This process is called amplification. In order to make all of these copies I use an instrument called a thermal cycler and a method called the polymerase chain reaction. At the end of amplification I have millions of copies of the 13 genetic markers for each DNA sample. The STR markers are tagged with different colored dyes. I will show you why that is important in a few slides.

Polymerase Chain Reaction (PCR) Here is a diagram showing the polymerase chain reaction. On the left in red is one of the genetic markers that I’m interested in. You can see here that each time I copy the genetic marker my numbers double so that by the end I have millions of copies of each genetic marker. This is done for each genetic marker.

Applied Biosystems 3130 Genetic Analyzer After I have amplified the genetic markers for each sample, I run the samples on an instrument like this one. This instrument is called a genetic analyzer.

3130 is a 4 capillary instrument Here is a closer view inside the instrument. Let me point out a few things. This is a tray that holds the DNA samples. (Use screen pen) to draw over capillary – and this is a capillary, which is like a very thin glass straw. It is slightly larger in diameter than your hair. The DNA samples travel though the capillary. Over here behind this black square is a laser. The laser is what is going to read the DNA samples as they pass by it.

DNA Analysis-hours at the computer!

Identifiler- 15 loci and amelogenin Here is an example of what the data looks like after it has been analyzed. Each of these areas with the gray bars is a different genetic marker. You can see that the genetic markers are tagged with different colored dyes. Notice that for some of the genetic markers you can see two peaks and at some of the other markers you only see one peak. Let’s look at one of the genetic markers that has two peaks. Remember Mom and Dad. So one of these alleles (peaks) came from Mom and the other allele came from Dad. Now let’s look at one of the genetic markers with only one peak. What this means is that Mom and Dad passed on the same allele for this genetic marker. You can see here that nine genetic markers are shown (not counting the marker for gender). We use a kit called Profiler Plus to copy these nine genetic markers.

Identifiler- 15 loci and amelogenin Here is an example of what the data looks like after it has been analyzed. Each of these areas with the gray bars is a different genetic marker. You can see that the genetic markers are tagged with different colored dyes. Notice that for some of the genetic markers you can see two peaks and at some of the other markers you only see one peak. Let’s look at one of the genetic markers that has two peaks. Remember Mom and Dad. So one of these alleles (peaks) came from Mom and the other allele came from Dad. Now let’s look at one of the genetic markers with only one peak. What this means is that Mom and Dad passed on the same allele for this genetic marker. You can see here that nine genetic markers are shown (not counting the marker for gender). We use a kit called Profiler Plus to copy these nine genetic markers.

Identifiler Tables

IDENTIFILER LOCI

Analysis continued A report with a table of results is generated The laboratory work and report are technically reviewed by a qualified analyst. The laboratory work and report are administratively reviewed. Report is released.

Comparison of Genetic Profiles Possible Outcomes of comparison of DNA Results to Known Reference DNA profiles Inclusion (reference profile matches evidence profile) Exclusion (reference profile does not match evidence profile) Inconclusive (not enough information to make comparison) After all of the data is analyzed I compare the profiles that I got for the reference samples to the profiles for the evidence samples to see if any of the samples match. There are three possible outcomes of this comparison. Inclusion (all of the alleles at each genetic marker match) Exclusion (if an allele at any genetic marker doesn’t match then this is an exclusion) Inconclusive (usually this is because the evidence sample is degraded)

Matching results from comparison What is the significance of a match between the reference profile and the evidence profile? Another way to think about this question is: How rare is the evidence profile in the population? If the genetic profile for a reference sample matches the genetic profile for an evidence sample then I need to find out what that means. Another way to think about this question is: How rare is the evidence profile in the population.

How rare is the evidence profile? First: Population Study A study that counts how many times a certain allele is seen for a genetic maker. The published data from the FBI is used for the 3 major racial/ethnic groups (Caucasian, African American and Hispanic). Before I can answer this question I need a couple of things. First, I need a population study. A population study counts how many times a certain allele is seen for a genetic marker. Usually multiple genetic markers are studied all at the same time. My laboratory uses the published data from the FBI for the 3 major racial/ethnic groups

STR Allele Frequencies 5 10 15 20 25 30 35 40 45 6 7 8 9 9.3 TH01 Marker Number of repeats Percent Caucasians (N=427) Blacks (N=414) Hispanics (N=414) Here is an example of the population data for one of the genetic markers. This STR marker is on chromosome 2. This chart shows the common alleles for this marker. If I look at the 7 allele you can see that for the Afican American population a little more than 40% have a 7 allele for this genetic marker. For the Caucasian population about 18% have a 7 allele. And for Hispanics approx. 34% have a 7 allele. *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Conveying Frequency A dice is six-sided. Therefore one in six= 1/6=0.1667. A 20-car parking lot full of cars, how many are blue? 5, 5 in 20= 5/20= 0.25

How rare is the evidence profile? Second: Apply Statisti Multiply the allele frequencies for each genetic marker. D3S1358 Now that I know the frequency for each of the alleles in my genetic profile I need to apply a statistical formula to figure out how rare the profile is. The first thing is to multiply the allele frequencies for each genetic marker. Evidence 15 , 16 .24631 or 25% X .23153 or 23% = .11 (in Caucasians)

How rare is the evidence profile? Second: Apply Statistics Multiply the frequencies for each genetic marker together to determine the overall frequency for entire the profile. D3S1358 VWA FGA D8S1179 Because each type is independently inherited, we can apply the product rule. We multiply the frequencies of all the genetic markers together to come up with an overall frequency for the entire profile. The average discrimination of the PCR Identifiler kit is see next slide. 15, 16 16, 18 22,25 13, 15 .11 X .09 X .03 X .07 1 in # quadrillion, 15 zeros Frequencies of 11 more genetic markers =

Discrimination power of Identifiler

Population of Calif. and USA CA is 36.7 million USA is 304 million

World Population ~ 6.79 Billion

Conclusion The evidence is presented and the strength of the match conveyed. We leave the decision of guilt or innocence for the jury to decide.