Str Multiplex Example

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An example of DNA profiles obtained from two different individuals using STR markers is shown in Figure 1.3. In a single amplification reaction, unique sites

Can a simple DNA test have the power to impact world events? In 1998, DNA evidence and independent counsel Kenneth Starr was investigating allegations that U.S. Monica Lewinsky's

President William Jefferson Clinton had a sexual relationship with a young blue dress

White House intern, Monica Lewinsky. President Clinton had publicly denied the allegations quite emphatically and at that time there was no concrete evidence to the contrary.

During the course of the investigation, a dark blue dress belonging to Monica Lewinsky was brought to the FBI Laboratory for examination. Semen was identified on evidence item Q3243, as the dress was cataloged. The unknown semen stain was quickly examined with seven RFLP single locus probes. Late on the evening of 3 August 1998, a reference blood sample was drawn from President Clinton for comparison purposes (Woodward 1999).

As in the O.J. Simpson case (see D.N.A. Box 3.2), conventional RFLP markers were used to match the sample of President Clinton's blood to the semen stain on Monica Lewinski's dress. At the time these samples were run in the FBI Laboratory (early August 1998), STR typing methods were being validated but were not yet in routine use within the FBI's DNA Analysis Unit. High molecular weight DNA from the semen stain (FBI specimen Q3243-1) and President Clinton's blood (FBI specimen K39) was digested with the restriction enzyme HaeIII. A seven-probe match was obtained at all seven RFLP loci examined.

This match was reported in the following manner: 'Based on the results of these seven genetic loci, specimen K39 (CLINTON) is the source of the DNA obtained from specimen Q3243-1, to a reasonable degree of scientific certainty.' The random match probability was calculated to be on the order of 1 in 7.8 trillion when compared to a Caucasian population database.

When faced with this indisputable DNA evidence, President Clinton found himself in a tight spot. Earlier statements that he had not had 'sexual relations' with Miss Lewinsky were now in doubt. The DNA results along with other evidence and testimony resulted in the impeachment of President Clinton on 19 December 1998 - only the second President in U.S. history to be impeached. This physical evidence played an important role in demonstrating that a sexual relationship had existed between Miss Lewinsky and President William Jefferson Clinton. Although during the Senate impeachment trial, it was determined that his deeds were not serious enough for him to be removed from office, President Clinton's career will always be tainted by the semen stain on the now famous blue dress.


Woodward, B. (1999) Shadow: Five Presidents and the Legacy of Watergate.

New York: Simon & Schuster. Grunwald, L. and Adler, S.J. (eds) (1999) Letters of the Century: America

1900-1999, p. 673. New York: The Dial Press.

on ten different chromosomes were probed with this DNA test to provide a random match probability of approximately 1 in 3 trillion. Note that every single site tested produces a different result between these two DNA samples. For example, marker A has two peaks in the top panel and only one peak in the bottom panel. Likewise, marker J produces two peaks in both samples but they result in different patterns due to different sizes at the site measured in the two DNA samples. These results can be reliably obtained in as little as a few hours from a very small drop of blood or bloodstain.

Each STR allele is distinguished from the others in the amplification reaction by separating it based on its length and color. The color results from a fluorescent dye that is attached during the amplification reaction. In this example, DNA markers B, E, H, and J are labeled with a blue colored dye, markers A, D, and G are labeled with a yellow dye, and markers C, F, I, and the gender ID are labeled in green. The gender ID results in two peaks for a male sample (X,Y) and a single peak for a female sample (X,X). Chapter 5 will describe the identity of the DNA markers represented in Figure 1.3.

Figure 1.3

Comparison of the DNA profiles for two individuals obtained with multiple short tandem repeat markers. STR length variation at unique sites on 10 different chromosomes are probed with this DNA test to provide a random match probability of approximately 1 in 3 trillion. A gender identification test also indicates that the top sample is from a male while the bottom sample is from a female individual. These results were obtained from a spot of blood the size of a pin head in less than five hours. The DNA size range in base pairs is shown across the top of the plot. Results from each DNA marker are indicated by the letters A—J.


In order to get a better feel for how rapidly forensic DNA analysis methods have progressed in the last two decades, a comparison to computer technology may be helpful. The use of computers at home and in the workplace has increased dramatically since personal computers became available in the mid-1980s.

100 125 150 175 200 225 250 275 300 325

100 125 150 175 200 225 250 275 300 325















These computers get faster and more powerful every year. It is almost inconceivable that the Internet, which has such a large impact on our daily lives, was just an idea a few years ago.

When multi-locus RFLP probes were first reported in 1985, the average computer operating speed was less than 25 MHz. Almost 20 years later in the year 2004, computing speeds of 2500 MHz (2.5 GHz) are now common. Computer processing speeds and capabilities have increased rapidly every year. Likewise, the ability of laboratories to perform DNA typing methods has improved dramatically along a similar timeline due to rapid progress in the areas of biology, technology, and understanding of genetic theories. In addition, the power of discrimination for DNA tests has steadily increased in the late 1990s (see Table 5.3, Table 20.8).

Some interesting parallels can be drawn between the Microsoft Corporation, the company that has led the computer technology revolution, and the timing for advancements in the field of forensic DNA typing (Table 1.1). In 1985, the


Forensic DNA Science & Application

Parallel Developments in Biotechnology

Microsoft Corporation Chronology


Alec Jeffreys develops multi-locus RFLP probes

PCR process first described

First version of Windows shipped


DNA testing goes public with Cellmark and Lifecodes in United States

Automated DNA sequencing with 4-colors first described

Microsoft goes public


FBI begins DNA casework with single-locus RFLP probes


TWGDAM established; NY u. Castro case raises issues over quality assurance of laboratories

DNA detection by gel silver-staining, slot blot, and reverse dot blots first described


Population statistics used with RFLP methods are questioned; PCR methods start with DQA1

Human Genome Project begins with goal to map all human genes

Windows 3.0 released (quality problems); exceeds $1 billion in sales


Fluorescent STR markers first described; Chelex extraction

Windows 3.1 released


NRC I Report; FBI starts casework with PCR-DQA1

Capillary arrays first described


First STR kit available; sex-typing (amelogenin) developed

First STR results with CE


Congress authorizes money for upgrading state forensic labs; 'DNA wars' declared over; FBI starts casework with PCR-PM

Hitachi FMBIO and Molecular Dynamics gel scanners; first DNA results on microchip CE

Major historical events in forensic DNA typing shown by year. The events relating to forensic DNA (first column) are described in context with parallel developments in biotechnology (second column) and key events relating to Microsoft Corporation, which have impacted the computer age (final column).


Forensic DNA Science & Application

Parallel Developments in Biotechnology

Microsoft Corporation Chronology


O.J. Simpson saga makes public more aware of DNA; DNA Advisory Board setup; UK DNA Database established; FBI starts using D1S80/amelogenin

ABI 310 Genetic Analyzer and TaqGold DNA polymerase introduced

Windows 95 released


NRC II Report; FBI starts mtDNA testing; first multiplex STR kits become available

STR results with MALDI-TOF and GeneChip mtDNA results demonstrated


13 core STR loci defined; Y-chromosome STRs described

Internet Explorer begins overtaking Netscape


FBI launches national Combined DNA Index System; Thomas Jefferson and Bill Clinton implicated with DNA

2000 SNP hybridization chip described

Windows 98 released; antitrust trial with U.S. Justice Department begins


Multiplex STR kits are validated in numerous labs; FBI stops testing DQA1/PM/D1S80

ABI 3700 96-capillary array for high-throughput DNA analysis; chromosome 22 fully sequenced


FBI and other labs stop running RFLP cases and convert to multiplex STRs; PowerPlex 16 kit enables first single amplification of CODIS STRs

First copy of human genome completed

Bill Gates steps down as Microsoft CEO; Windows 2000 released


Identifiler STR kit released with 5-dye chemistry; first Y-STR kit becomes available

ABI 3100 Genetic Analyzer introduced

Windows XP released


FBI mtDNA population database released; Y-STR 20plex published

Windows XP Tablet PC Edition released


U.S. DNA database (NDIS) exceeds 1 million convicted offender profiles; the UK National DNA Database passes the 2 million sample mark

Human Genome Project completed with the 'final' sequence coinciding with 50th anniversary of Watson-Crick DNA discovery

Windows Server 2003 released; 64-Bit Operating Systems expand capabilities of software

Table 1.1 (Continued)

year that Alec Jeffreys first published his work with multi-locus RFLP probes, Microsoft shipped its first version of Windows software to serve as a computer operating system. In 1986, as DNA testing began to 'go public' in the United States with Cellmark and Lifecodes performing multi-locus RFLP, Microsoft went public with a successful initial public offering.

In the late 1980s, single-locus RFLP probes began to be used by the FBI Laboratory in DNA casework. Due to issues over the use of statistics for population genetics and the quality of results obtained in forensic laboratories, RFLP methods were questioned by the legal community in 1989 and the early 1990s. At this same time, Microsoft had quality problems of their own with the

Table 1.1 (Continued)

Windows 3.0 operating system. However, they 'turned the corner' with their product release of Windows 3.1 in 1991. In the same year, improved methods for DNA typing were introduced, namely fluorescent STR markers and Chelex extraction.

The popularity of Microsoft products improved in 1995 with the release of Windows 95. During this same year, forensic DNA methods gained public exposure and popularity due to the O.J. Simpson trial. The United Kingdom also launched a National DNA Database that has revolutionized the use of DNA as an investigative tool. The United States launched their national Combined DNA Index System (CODIS) in 1998, concurrent with the release of Windows 98.

To aid sample throughput and processing speed, the FBI Laboratory and many other forensic labs have stopped running RFLP cases as of the year 2000. On 13 January 2000, Bill Gates stepped down as the CEO of Microsoft in order to help his company move into new directions.

The development and release of Windows 2000 and Windows XP at the beginning of the 21st century continue to improve the capabilities of multitasking computer software. In like manner, the development and release of new DNA testing kits capable of single amplification reactions for examining 16 regions of the human genome furthers the capability of multiplexing DNA information (see Chapter 5).

We recognize that due to the rapid advances in the field of forensic DNA typing, some aspects of this book may be out of date by the time it is published, much like a computer is no longer the latest model by the time it is purchased. However, a reader should be able to gain a fundamental understanding of forensic DNA typing from the following pages. While we cannot predict the future with certainty, short tandem repeat DNA markers have had and will continue to have an important role to play in forensic DNA typing due to their use in DNA databases.

The match on Mr. Davis described at the beginning of this chapter was made with eight STR markers. These eight STRs are a subset of 13 STR markers described in detail throughout this book that will most likely be used in DNA databases around the world for many years to come. Perhaps with odds of getting caught becoming greater than ever before, violent criminals like Mr. Davis will think twice before carrying out such heinous actions.

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