DNA, Semen Analysis, and Y-Chromosome Testing

     Yesterday Fox News released the following statement in a report about the Duke Lacrosse case: 
     "On April 8, 9 and 10, DNA Security found DNA from multiple males on the panties and from rape-kit swabs..... and that not only did the DNA not match the three defendants, but that it also did not belong to any of their lacrosse teammates or anyone else who submitted DNA samples"

     In light of that, I'd like to explain a bit about DNA, semen analysis and Y-Chromosome testing.

 

Basic DNA

     There are approximately 60 trillion cells in the human body. Each of these cells carry material called chromosomes. Along those chromosomes are about 100,000 genes. The gene is what carries our heredity.

   It determines everything from our eye and hair color to our risk for diseases like heart disease and diabetes.  Inside each gene is Deoxyribonucleic Acid - DNA - whose purpose is to carry out a single function.

   "DNA is a polymer,a molecule, made up of a large number of atoms, arranged in repeating units called nucleotides." ( Criminalistics, Saferstein, Richard, 2001) Nucleotides are then strung together to form thew DNA strand.

   There are four bases attached to the backbone of the DNA strand: adenine, cytosine, guanine, thymine. The DNA strand can go on forever containing millions of bases.

 

Double Helix

   There are actually two DNA strands called a double Helix. They resemble two wires twisted around each other.

   The two strands are connected by the four bases, adenine, cytosine, guanine, and thymine ( A,C,G,T).

   The pairing of the bases has to go in a certain pattern. Although there can be different sequences, A must attach to T and G must attach to C:

A........T

G........C

C........G
A........T

T........A
C........G

   This is called base pairing. Although this is the way the bases must be paired together, there is no limit on how the bases are sequenced on the strand. So there are limitless possibilities of sequencing.

   Each strand of DNA has the same information. It's how the bases are sequenced that determines our unique genetic heredity, including any predisposition to certain diseases.

 

All the human chromosomes contain about three billion base pairs. This is referred to as the genetic code. An individualized code that is as unique to each of us as our fingerprints, except in the case of identical twins.

 

DNA Analysis

   In order to analyze a person's DNA, it must first be replicated, then run through certain machines and processes. Replicating DNA starts with unwinding the DNA strands in the double helix. This is done in order to make new DNA from existing DNA.

   Then the double helix is re-created, pairing the bases A,C,G, and T correctly along the strands. The result is now two identical copies of the DNA strands instead of one.

   This process is called Polymerase Chain Reaction (PCR). Small quantities of DNA found at a crime scene can be replicated by copying the DNA strands in a machine called a DNA Thermal Cycler.

   Each cycle of of the PCR causes the DNA strand to be duplicated and within a matter of hours, DNA can be duplicated a million fold.  Once the scientists have enough DNA copies on hand, they can then analyze them.

DNA Typing

     Each chromosome has two DNA double helix strands and contains the repeating base sequence, T - A - G. These repeating sequences are inherited from our parents. One parent might contribute two repeating sequences, another four.

   How many times the base sequences are repeated is the key to our hereditary factors. With the technique, restriction fragment length polymorphisms (RFLP), enzymes are used to cut up the chromosomes into multiple fragments, some containing the repeated sequences.

   Once that's done, the fragments are then separated by electrophoresis. DNA is then put in a gel and placed in an electric field.

   During electrophoresis, the DNA fragments will migrate across the plate. The smaller fragments will move faster than the slower ones. Once this is done, the strands are chemically treated so they stay separated.

   The strands are then transferred to a nylon material, which is treated radioactively in order to visualize them.

   Next the nylon sheet is exposed by x-ray for several days. Afterwords, visible bands show up.(Saferstein) The bands sets are then compared to see if they match up.

   For example, comparing a suspect semen stain, taken from a crime scene, to the victim's blood.

   The RFLP has since been replaced by newer techniques, which are more sensitive and require less DNA material. The PCR is able to use only one billionth of a gram for analysis and only needs tiny quantities of body fluids, such as blood, saliva, or semen.

   Another advantage is that PCR can analyze even degraded samples which RFLP couldn't do. PCR can duplicate a DNA strand many millions of times in a short period of time.

Short Tandem Repeats (STR)

   " STR's are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule."

   These strand are much shorter than those used in the RFLP procedure and are less susceptible to degradation, so they can be taken from decomposed bodies.

   "The ability to copy an STR means that extremely small amounts of the molecule can be detected and analyzed." The human genes contain hundreds of different types of STR's.

   The more STR"s present, the smaller the population which can carry them, and the greater the probability of identification.

Nuclear & Mitochondrial DNA

      A human cell contains two types of DNA: Nuclear and Mitochondrial. Nuclear DNA has 23 pairs of chromosomes within the nuclei of the cell. Each parent contributes to half the genetic make up of the chromosomes.

   Mitochondria DNA is inherited only from the mother and is found outside the nucleus of the cell, as opposed to the one nuclear DNA within the nucleus of the cell.

   Each cell contains hundreds of thousands of mitochondria and one nuclear DNA. Mitochondria (MtDNA) DNA is helpful if forensic scientists have degraded or skeleton specimens, or specimens with a low nuclear DNA. However, MtDNA is more costly and time consuming to analyze.

 

Collection of Biological Evidence

      Since the early 1990's, DNA has has changed the way the criminal justice system looks at biological evidence. In the past, crime labs were able only to do an ABO typing from blood. Now with one billionth of a gram of DNA, they are able to identify "genetic fingerprints".

      If readers will check my archives, you'll find Crime Scene Investigation, which explains the collection of evidence, so I won't repeat that here.

Two Victims

     In 1982, a woman was raped in a park, as she walked away from her husband, heading to their car.The woman was white and she said the offender was black.

   Vincent Jenkins, a 43 year old black man, was picked up four months later. The victim was asked to identify the accused via a one way mirror.

   She said she couldn't identify him as the man who had raped her. Then the police showed her a four year old photo and said it was a current picture of the man.

She looked at it for a long time, then said that he was the man who had raped her. The court ruled that the photo identification was "unconstitutionally suggestive", so the evidence was excluded from trial.

    The jury still convicted Jenkins, even though the physical description differed widely from the accuser's initial description. Jenkins was convicted and served almost 17 years in jail before being exonerated by newer DNA testing methods.

   The new testing showed a surprise. The testing showed two different sets of DNA. However, It not only cleared Jenkins, it also excluded the victim's husband. Even after being cleared by the DNA, the DA fought the release.

   The prosecutor tried to develop different hypotheses to explain why Jenkins's DNA was excluded, yet he could still be the rapist. The prosecution came up with a gang rape theory, in which three rapists were involved.

      " Those hypotheses, born of desperation, are contradicted by the victim, who said ... that she had been raped by one man and that he had ejaculated." According to the DNA, Jenkins was not that man.

Bob Herbert, NYT: "DNA tests have ruled out Mr. Jenkins as the man who attacked and raped a woman in a nature preserve in Buffalo in 1982.  But it is easier to learn to fly by flapping one's arms than it is to get a district attorney to admit having sent an innocent person to prison. "

Vincent Jenkins was finally freed in September, 1999. I can't help but wonder what he would have to say about being falsely accused and convicted.

* Story originally published 1999 in NYT

 

Forensic Characterization of Semen

     The average male ejaculate contains anywhere from  2 - 6 milliliters of seminal fluid and about 100 million sperm. In order to analyze semen, the evidence must first be determined to be semen. This involves two steps:

Acid Phosphatase

    Acid phosphatase is an enzyme that secreted in seminal fluid, as well as other bodily fluids, such as vaginal secretions. However, the concentration of Acid Phosphatase is up to 400 times greater in semen than in any other body fluid.

   It can be detected by its reaction to a special acidic chemical compound. A quick purple color is indicative of a positive acid phosphatase reading.

   There are some fruit and vegetable juices which also have acid phosphatase, but they will not react as quickly as Acid Phosphatase in seminal fluid. A reaction time of less than 30 seconds indicates (+) semen.

  Another absolute indicator of semen is sperm. You can have semen without sperm, but you can't have sperm without semen. When sperm are located under the microscope, a stain is definitely (+) for semen.

  Sperm are long, with both a head and a tail. The head carries the genetic material and the tail helps to spur it along upstream towards the cervix. Even with the large number of sperm in seminal fluid, it's not unusual to have difficulty spotting them under the microscope.

   Not only are sperm bound tightly to cloth but, once dried, they become very brittle and easily disintegrate when washed or rubbed against something.

     Another reason is that sexual assaults often involve men with an abnormally low sperm count. This is known as Oligospermia. Some offenders have no sperm count at all. This is called Aspermia.

   A third reason is the increasing use of condoms by the offenders. They are getting smarter. Until last year, the nurses in my unit took swabbings for a sperm check down to the lab after each exam. It was rare that any sperm showed up under the microscope.

   If no sperm can be detected, yet the scientists still believe the evidence to be semen, a test known as the Prostate Specific Antigen (PSA or P30) can be carried out. This test is specific to seminal plasma.

   When it is isolated an injected into a rabbit, it produces antibodies ( anti-P30).

   The serum is then used to check the semen stain. If it tests positive for P30, then the stain is seminal. Once the evidence is proven to be semen, the next step is to link the evidence to an individual through DNA fingerprinting.

Collection and Dating Evidence

      Saferstein states, " The finding of seminal constituents ... is important evidence for substantiating that sexual intercourse has taken place ... but their absence does not necessarily mean that a rape did not occur."  He goes on to say, " Physical injuries such as bruises or bleeding tend to confirm that a violent assault did take place.

   Furthermore, there is a distinct possibility that the forceful contact between victim and assailant will result in a transfer of physical evidence." 

   To protect the evidence, all garments and evidence must be collected according to strict protocols.

     "The persistence of seminal constituents in the vagina may become a factor when trying to ascertain the time of an alleged sexual attack."

   It's important to note that motile sperm only live up to 4-6 hours in the vaginal canal. The acidic environment of the vagina decreases the motile sperm count rapidly.

   Non motile sperm may normally be found up to 3 days after intercourse, and sometimes up to 6 days later in the cervix. Intact sperm (with tails) aren't usually found after 16 hours post intercourse but have been found up to 72 hours.

   Seminal Acid Phosphatase substantially decreases with time in the vagina. It's not usually found in the vagina after 48 hours. Another indicator of recent sexual activity is the P30 test. P30 is not usually found in the vagina after 8-9 hours post intercourse.

   As far as the Duke case goes, I realized this morning that I had made a mistake in judgment. For some reason, I had thought that semen had been found originally. If semen had been found, that would indicate a (+) P30 test and P30 only lasts in the vaginal canal about 8 hours; Acid Phosphatase lasting only 48 hours.

   Those results would indicate a much more recent sexual activity than the accuser stated. According to the motion filed by the defense, that is not true. However, there is more to that which will be discussed below. I believe the findings by the crime lab reported last night are even better for the Duke Lacrosse players.

   If there was complete ejaculation, as the accuser claimed, then there should have been high levels of Acid Phosphatase and P30, and possibly motile sperm, left by the offenders, in her vagina and on her underwear. 

    A few years back, I kept close tabs on the Kobe Bryant case as well. I especially remembered when the scientist Dr. Elizabeth Johnson testified. I even took notes.

   I'm including a link to that testimony. In her testimony, Dr. Johnson testifies specifically as to how much Acid Phosphatase, P30, and sperm should still be present after a recent assault.

      I find it very interesting, considering that such a vicious attack (with ejaculation)supposedly occurred, and a SANE exam was done shortly afterwords, how come no Acid Phosphatase, no P30, and no sperm is connected back to any of the three Duke players charged?

   Excerpt, Dr. Johnson's Testimony:

  "1 was ejaculation internally or not. I mean, we've seen 2 plenty of instances in case work where there was penetration 3 without ejaculation, we don't find any or many sperm. We 4 don't find P30. It depends on how the sex occurs. I mean, 5 it can be very -- 6
   Q. But if there is ejaculation, then generally you're 7 going to find a lot of sperm? 8 A. If it's internal, yes. If it's a full ejaculate 9 internal, yes. If it's partial, no. If it's just 10 penetration, few to none. 11 Q. Now, what exactly is P30? 12 A. P30 is a protein that is found, is a prostate -- 13 thought to be a prostate specific protein produced by the 14 prostate gland produced in high levels in the seminal fluid.
     15 Q. Are these only this males, then? 16 A. There have been a few studies using a very 17 sensitive radioimmunassay test in which that has been found 18 in some fluids of urine of men that have prostate cancer or 19 breast milk from some women, even, but it's a different type 20 of test they are employing than what we are using here. 21 Q. So if, I mean, for your testing it is basically a 22 male -- it comes from the male, P30, for purposes of your 23 testing? 24 A. For the purposes, yes, here."

Y-Chromosome Testing

     This is among the newest of the testing procedures. It's fast and needs little DNA. Since this test is specific for only the male Y chromosome, it's very useful for determining the number of male donors, in a mixed specimen, such as with a sexual assault. It can be tested on sperm as well as epithelial cells.

    "We have exemplified the potential utility of MPI and MPII in operational casework by their ability to dissect out the full 19 locus male haplotype in vaginal swabs taken up to 48 hours after intercourse. Partial profiles can be obtained 85 hours after intercourse. A judiciously chosen eleven-locus subset of our nineteen Y-STR loci is able to obtain a male donor profile 4 days after intercourse. "

     Again, they have developed the ability to determine recent sexual intercourse. As stated above, male halotypes can be extracted within just 48 hours post intercourse. This is important in cases such as the Duke Lacrosse case when the evidence, once again, appears to contradict the accuser's statements.

 

 

 
   
 

 

   

      


   

   

Comments

[Jason]

This article states that "Along those chromosomes are about 100,000 genes." This is wrong and should be corrected. There are actually only around 25,000 genes.

[Kathleen]

Jason:
Richard Saferstein's authoratative textbook, Criminalistics. 7th Ed., 2001, Pg. 354, states, " Inside each of 60 trillion cells are ... chromosomes..." and that "there are nearly 100,000 genes... arranged along the chromosomes."

[Rohan sugathadasa]

Dear sir,
Please be kind enough to send me the protocol for DNA, Semen Analysis, and Y-Chromosome Testing.

Thanks

[Dr. Geetha Lakshmi.P.]

Sir,
I am working as a forensic scientist in India. I want to know is there any preliminary tests- such as microscopic or chemical tests are available to compare the spermatoza available from accused and victim, i.e., can we determine they come from the same person?
Geetha Lakshmi.P.

[abed]

dear sir
I am working in forensic scientist in iran .Please be kind enough to send me the protocol for DNA, Semen Analysis, and Y-Chromosome Testing
tank you

[Robert]

In 1986 I was beaten to sign a false confession in a sex crime. The only cooberative evidence at trial was that they did (within an hour of alleged incident) an acid phosphotase test which turned light blue, then they sent the swab to the State Police lab and the Doctor testified that he saw under a microscope "very few heads of sperm and no semen". I had the swab tested for DNA several years later and the results were that there was nothing on the swab at all. The District Attorneys' position after that test was "Well it is our position that it (the sperm head}was there when we looked at it the first time. This case is long over but I am interested in finding a scientist to discuss the case with in my petition for removal from sex offender registry requirements, and a posible pardon from the govenor.