This paper was last updated on Thursday March 08, 2012

DNA is made up of four components Adenine, Cytosine, Guanine, and Thymine. These four components of DNA form microscopic coiled structures called chromosomes. Most people of 23 pairs of these and they inherit one chromosome from their father and the other from their mother. Together, these chromosomes are used to create genes determined by the order of the four component bases (ACGT) and their location or placement (locus).  Any form of a gene that can occur in a particular locus is called an allele. Some genes have as many as several hundred different alleles. In general, people inherit two alleles, one from their mother and one from their father. Only rarely, both alleles at a particular locus come from one parent but sometimes it does happen.

Mitochondrial DNA is only inherited from the mother and is located in the mitochondria outside the cell nucleus. Nuclear DNA occurs in pairs where one copy is inherited from the genetic father and the other is inherited from the genetic mother. The genetic composition of humans consists of more than three billion base pairs of nuclear DNA and approximately 16,569 base pairs of mitochondrial DNA.

Sometimes the amount of biological evidence that may be recovered is so small that there can be questions about testing those samples using low count DNA testing. Since some who read this paper may not have a sense of the magnitude of the range of sample sizes, I have provided a comparison of the unit of measures in the table below. Low count DNA testing involves the testing of genetic material in the picogram range.

The measure of any testing standard is its reproducibility, the ability to reproduce the exact result each time. Moreover, the result must be useful. If the test result cannot be reproduced each time then the test or testing procedure is neither useful nor reproducible but useless. Nevertheless, these limitations help define the limits of the techniques and the technology to be used and that is an important consideration. It is better to know at what point where a process cannot produce consistently reliable results. This helps determine the limits of testing AND to be able to save a portion of the sample for future testing when the technology has improved.

Control samples are used to define and validate the limits of the techniques being used in the laboratory. The methodology may include a progressive dilution study to determine at what specific point the detection mechanisms cannot produce consistent and reliable results. This is important because some samples are so small that they must be tested by a facility that can do those tests. Otherwise, there would likely be no remaining material to be tested. This leads to knowing the effects of amplification and knowing when to stop.

Stochastic changes are random and that means that they a result of chance and probability - or some other unforseen factor that affects an experiment or a person’s choices. For an example, the term ‘forensic’ is the study of argumentative discussion but it also includes the analysis of all of the physical evidence, biological evidence, genetic evidence, and other information.

So the problem is recognizing and distinguishing stochastic (random) variations from those that either occurred normally, or through improper facilities or procedures, or deliberate adulteration. So an aliquot does not have to have a specific size, it is merely a fractional portion of a solution such that the dilution remains the same. The advantage here is that aliquots can be replicated to produce DNA control samples. These control samples then can be used to establish the point of dilution where a detection method fails to produce consistent results such that it is the benchmark that leads to ‘reproducibility’.

Stochastic Effects

A stochastic effect is simply a random variable that occurs while attempting to recover small amounts of DNA from a sample. Another way of explaining part of this effect is that the sample can include junk material with the DNA during the early cycles of amplification. This means that refining process must remove the unwanted material from the sample or tagged in some way to distinguish them from the DNA of interest. So the next problem is that the primers used to amplify a specific location might not find and refine all of the targeted molecules that are present in the sample.

Moreover, with a heterozygous locus (two alleles at the same place) unequal or inconsistent sampling can result in the failure to detect one or both alleles. So researchers call the loss of a single allele an ‘allele dropout’ and they call the loss of both alleles a ‘locus dropout’. The catch here is that amplifying the same DNA extract twice, can result in the detection of different alleles at a specific locus. So how are these random effects avoided?

One method is to stop testing before reaching the stochastic realm but the problem is how can anyone know when they are too low to get consistent and reliable results? One way of doing this is by relying upon experience and using established interpretation guidelines that are based upon prior validation studies. In other words, you follow the trail that you or others have established.  So another method is to increase the number of PCR cycles to get as much genetic data that process can obtain from the sample. This presents another problem because continued testing of a limited amount of genetic material could consume it.  This introduces another issue.

How is it possible to amplify a specific strand of DNA in a mixture without amplifying other DNA that may remain, and used later? The other part of the issue is this, how did a person’s DNA become a part of the mixture? If a grain of sand from the Sahara reached a home in Alaska, what a long journey would have that been? The problem is knowing when to stop and avoid introducing unwanted and misleading artifacts.

Recording DNA Signatures

Researchers often use charts to record information. Unfortunately many people use the term ‘chart’ when they mean a folder (that can contain charts). The kind of chart that I have in mind has a zero baseline because no quantity can be less than zero and the actual markings used on the chart resemble very skinny isosceles triangles. They appear as spikes of various heights and these spikes represent the relative amount of genetic material at a specific locus.

 Unlike other methods, DNA presentations do not have a negative region and this means that DNA is either is or is not present. That is only in a perfect world but we do not live in a perfect world. So when testing increases the number of PCR cycles, these anomalies become more pronounced and we call these stochastic anomalies.  Four stochastic anomalies could occur during the amplification of extremely low amounts of DNA. One important consideration is that amplifying the same DNA extract more than once, can result in the detection of different alleles at the same location. That is the next topic in this presentation.

Detecting Mixtures and Anomalies

A wild card is designation for an unknown factor and it is supposed to be an arbitrary guess for a missing factor. In the early grades of elementary school, this technique was often used to teach students in matters of arithmetic by filling the blank space with the correct value. The problem with wild card designations can be illustrated by the mythical accountant’s reply to his boss about two plus two and its answer. What do you want it to be? So the problem is how does one avoid garbage in, garbage out (GIGO)?  How does someone create a stable amplification that will only produce a pure sample of the DNA of interest without unwanted artifacts?

In DNA testing, extremely small amounts of DNA can be detected by increasing the sensitivity. This has a cost because the ideal process will only amplify the DNA of interest while increasing its purity. In other words, the perfect amplification will only produce a pure sample without unwanted artifacts and this requires an inherently stable process that can be easily controlled.

So the requirement for the ‘perfect’ DNA refinery is that will only amplify selected DNA, the process can be easily controlled, and it will not introduce nor create impurities. An allele is an alternate form of a specific gene at a particular locus (location). So people can be distinguished by either the presence or absence of a specific allele in their DNA.

Mixtures are probably the most common occurring problem because there may be more than one contributor to sample. Here, the display (or recording) device will show three or more alleles at the same place (loci). Two alleles will belong to the prime contributor and at least one other will belong to the other contributor. Remember that an allele is an alternate form of a specific gene at a particular locus (location). So people can be distinguished by the presence or absence of a specific allele in their DNA.

Wild Card Designations

Peak height imbalances result when there is at least a 30 percent difference between the height of two peaks. A person’s sample should contain peaks that are roughly the same height. Otherwise, the difference in these peaks is usually the result of a mixture between two or more sources.  Mixtures are probably the most common occurring problem because there may be more than one contributor to sample. So the recording device may show several alleles at the same place (loci) and so the depiction of several alleles may indicate that the sample contains DNA that belongs to one or more contributors.  A person’s sample should contain peaks that are nearly the same height.  Otherwise, the difference in these peaks is usually the result of a mixture between two or more sources.

Degradation is easily visualized as a set of falling peaks heights. Degradation can be hard to interpret because degraded samples often subject to allelic dropouts. This means that the process of degradation may lead to incomplete and inaccurate results because of allelic transformation.  The caveat is that stutter-peaks, tiny peaks that occur before or after the actual peak, are artifacts of the process rather than an actual indicator. Degradation is easily seen as a set of falling peak heights that can be confused with allelic dropouts. A geometric slope detection routine in a computer program or a simple straight edge can help determine deference between the ‘pq’ genotype (multiple alleles) and the ‘pp’ genotype (no alleles). Wild card designations such as ‘px’, where ‘x’ is unknown lead to false inclusions because they are what someone wants ‘x’ to be.

Simplified Molecular Chemistry

An atom is the smallest particle of an element. The smallest atom is that of hydrogen and it consists of only one proton and one electron. A proton has a positive charge while an electron has a negative charge. All atoms have a neutral charge because they have equal numbers of electrons and protons. An ion of an atom or a group of atoms has a positive charge if it has lost one or more electrons and has a negative charge if it has gained one or more electrons. Free electrons and free protons are also called ions because they are charged subatomic particles.

Hydrogen is colorless, odorless, and is highly flammable. It is the simplest and lightest element and normally exists as a diatomic gas. This means that the hydrogen molecule as two hydrogen atoms. Hydrogen is also unique because it is the only element that does not have neutrons in its nucleus but hydrogen has two isotopes that do, deuterium and tritium. Deuterium has one proton and one neutron while the element tritium has one proton and two neutrons. The next element in the periodic table is helium and its atom consists of two electrons, two protons and two neutrons.

The elements are usually arranged according to their atomic number but that doesn’t always work well because researchers may have different uses and requirements for the same chart. That is why most charts are actually compromises. They are made so that a series of related elements with similar properties (especially the Noble elements) may appear together, after the appropriate spacing, according to their atomic number. This is why Helium, Neon, Argon, Krypton, Xenon, and Radon appear as they do in the Periodic Table.

These elements occur as single atoms instead of molecules of two or more atoms and they have stable arrangements of electrons. Each successive element in this series has an added proton and electron. The added proton brings the outer electrons closer to the nucleus while decreasing the atomic radius and the added electron increases ionization energy. This is why these six gaseous elements are called the ‘noble gases’ because they do not react easily with other elements. Krypton, xenon, and radon do combine with fluorine and oxygen to form molecules.

Atoms and molecules are the basic units of matter. While an atom is the smallest particle of an element, a molecule has one or more atoms and is the smallest particle that retains all of the properties of a particular substance. A molecule can be part of a mixture but a mixture cannot be a molecule. The reason for this is that if a molecule is divided further, then only the molecular fragments or the atoms of the elements would remain. For an example, if a water molecule were divided, then only the atoms of hydrogen and oxygen would remain but where did the energy binding go?

Most of Earth’s water comes from mineral-laded lakes, oceans, and seas and this makes water a good conductor. Even so, the electrolysis of pure water requires far more energy to overcome the initial ionization. Pure water has an electrical conductivity of about one-millionth of that of sea water or mineral water.  Water (H₂O) can be decomposed into hydrogen gas (H₂) and oxygen gas (O₂) by passing an electric current through water.

Then the gases can be captured by small test tubes filled with water. So in the electrolysis of water, the positive charged hydrogen ions will appear at the cathode (negative ‘-‘) and negative charged oxygen ions appear at the anode (positive ‘+’). The gases then can be captured by small test tubes filled with water. So the electrolysis of water will produce twice as much hydrogen than oxygen and the total rate of production will be proportional to the power (voltage x amperage) conducted by the solution. So the reaction is expressed by

2H₂O = H₃O⁺ _(aq.) + OH^- _(aq.) where H₃O⁺ _(aq.) is the Hydronium Ion.

The electrolysis of water will produce twice as much hydrogen than oxygen and the total rate of production will be proportional to the power (voltage x amperage) conducted by the solution. Water molecules are constantly changing and renewing their hydrogen bonds such that water can exist as a gas, liquid, and a solid simultaneously. This is called the ‘triple point’ of water and that point occurs at 0⁰ Celsius (32⁰ Fahrenheit). The (aq) designation is simply shorthand for a solution that contains water (aqueous).

Molecules are made up of atoms held together in certain arrangements. Scientists use chemical formulas to show the composition of molecules. For example, a water molecule consists of two hydrogen atoms and one oxygen atom, and it has the formula H₂O. So a molecule's size and shape depend on the size and number of its atoms and the binding forces between the atoms.

A molecule that consists of two atoms, such as nitric oxide (NO), is called a diatomic molecule while a molecule made up of three atoms, such as water, is called a triatomic molecule. Four atoms create a tetradic molecule while five atoms such as those found in methane create a tetrahedral atom (CH₄). At the other extreme, a DNA molecule can contain millions of atoms and this is just one property that makes DNA molecules unique. The other property that makes DNA molecules unique is their molecular mass.

Atoms form molecules through strong attractive forces called bonds and the shape of a molecule depends upon the size and position of the atoms in the molecule. So an isomer is one of two or more compounds, radicals, or ions that contain the same elements and the number of their atoms but are different in their structural arrangement and properties.

This means that atoms occupy positions to form the strongest bonds that a particular group of atoms could form while other atoms move apart. For an example, an ammonia molecule has the shape of a tetrahedron (a four-faced pyramid). It consists of three hydrogen atoms attached to a nitrogen atom. Butane molecules have four carbon atoms arranged in a zigzag chain with 10 hydrogen atoms attached while large protein molecules can form long spiral chains.

Isomers are two or more chemical compounds that have the same number and type of each kind of atom, but they differ in the way that their atoms are arranged. Since their atomic arrangement varies, isomers are generally different from each other in both their physical properties and their chemical behavior.  For example, normal butane and isobutane are isomers of butane because both have the same components (atoms) and formula but the arrangement of atoms in their molecules is different. Moreover, normal butane boils at -0.5 degrees Celsius while isobutane boils at -12 degrees Celsius.

Optical Isomers are molecules that have different three-dimensional placements such that they are mirror images of each other. Moreover, these particular molecules cannot be converted into another molecular arrangement. Optical isomers have the physical property of being able to be distinguished by how they rotate polarized light. Levorotary means left and dextrorotatory means right rotation. Sometimes only one optical isomer of a substance is active while the other optical isomer is not.

The mass (amount of matter) of a molecule is indicated by its relative molecular mass. You can find the relative molecular mass of a molecule by adding the relative atomic masses of all the atoms in the molecule. An atom's relative atomic mass equals its mass divided by 1/12 of the mass of an atom of carbon 12, the most abundant form of carbon.  Here is an example.

What is the relative molecular mass of a molecule of carbon dioxide (CO₂) that consists of one atom of carbon 12 and two atoms of the most abundant form of oxygen? The relative atomic mass of the carbon atom would be exactly 12, and the relative atomic mass of each of the oxygen atoms, rounded to five figures, would be 15.995. The calculation would be 12.000 + 15.995 + 15.995 = 43.990.  A molecule's mass can also be measured with an instrument called a mass spectrometer.

Polar Molecules and Ionic Substances

Each atom in a molecule consists of a positively charged nucleus surrounded by a cloud of negatively charged electrons. In a neutral molecule, the positive and negative charges are evenly balanced throughout the molecule. In polar molecules, the charges are not evenly balanced. In a polar molecule, more positive charge collects at one location in the molecule and more negative charge collects at a different location. Some molecules are magnetic because of the way the electrons are unevenly distributed within the molecule.

Most gases, most common liquids, and many solids are made up of neutral or polar molecules. But some substances are made up of units called ions (atoms or groups of atoms with a positive or a negative charge). These substances are called ionic substances.

Molecules are held together in a group by electrical forces called the "van der Waals" forces. These forces are usually weaker than those that hold a molecule itself together. The force between molecules depends on how far apart they are. When two molecules are widely separated, they attract each other. When they come very close together, they repel each other.

In a solid, the molecules are so arranged that the forces which attract and repel are balanced. The molecules vibrate about these positions of balance, but they do not have enough energy to move to different parts of the solid. As the temperature of a solid is raised, the molecules vibrate more strongly. So when the van der Waals forces can no longer hold the molecules in place, the solid melts. In a liquid, the molecules move about easily but they still exert attractive force on other molecules. These forces keep the liquid together and surface tension creates attractive forces that cause a liquid to assume a shape of that has the least surface area.

Certain organic compounds called liquid crystals have properties of both liquids and solids. Within a particular temperature range, these compounds flow like a liquid, but have a more orderly molecular arrangement. Its molecules line up side by side and form tiny groups or clusters that slide past one another in certain directions.  In a gas, the molecules move about so fast that their attractive forces have little on other molecules. When two gaseous molecules collide, their repelling force sends them apart. This is why gas molecules fill a container completely, because they move freely through all the space available.

Most substances can be changed into solids, liquids, or gases by either raising or lowering their temperatures. The temperature at which these changes occur and other characteristics of a substance depend on the size, shape, and mass of the molecules and the strength of the van der Waals forces between them.  Under certain conditions, two molecules may collide with enough energy to react and form one or more new molecules. The process by which many small molecules combine chemically to produce a large molecule is called polymerization. Molecules can also break down into smaller molecules. Causes of molecular disintegration include ultraviolet light, fast-moving electrons, and nuclear radiation.

Researchers also learn about molecules by studying the way they absorb or give off light. Each kind of molecule absorbs or gives off certain colors of light. This group of colors makes up the molecule's spectrum. By studying the spectrum of a substance, scientists can find the sizes and shapes of its molecules. Moreover, they can determine the strength of the forces that hold the molecular atoms together and the way the electrons move within the molecules. This is similar to studying the spectra of stars and what they contain. Each element in the periodic table can appear in gaseous form and will produce a series of bright lines that are unique to that element. This means that hydrogen will not look like helium nor will carbon appear as nitrogen. This is the science of spectroscopy.

Gel Electrophoresis

Electrophoresis refers to the movement of suspended and dispersed particles through a medium through the action of direct current electromotive force. This phenomenon was first reported by F.F. Reiss in 1807 but the actual theory of this process (electrophoresis) has been debated ever since.

Polymerase Chain Reaction produces a mixture of molecules that has to be resolved from each other. Since an allele is an alternate form of a gene at a given locus (position), how can two or more alleles can be distinguished from the other? This reminds me of the coin sorters that are still used in banks, stores, and in other places. The largest coin is the quarter, followed by the nickel, penny, and then the dime. So all the sorter needs are a sequence of plates with holes that only allow successively smaller coins to pass and this is called a sieve. So electrophoresis is a technique for separating organic substances (included DNA and RNA) by their size and electric charge.  Remember this, the most important issue is that the separation method must be reproducible so that the results can be compared and verified by other independent laboratories.

In the electrolysis of water, the positive charged hydrogen ions appear at the cathode that has a negative charge and the negative charged oxygen ions appear at the anode that has a positive charge. So opposite polarities attract the other while same polarities repel the other. This is also true with magnetic polarity.  So the process of electrophoresis is based on the electrical charges of the ionic molecules including those of DNA.

DNA phosphate groups have a negative charge because they readily release their H⁺ ions. So DNA molecules will move away from the cathode (the negative electrode) and move toward the anode (the positive electrode). The rate of travel from the cathode and to the anode is controlled by adjusting the voltage applied to the sample. The greater the electromotive force (the voltage) the faster the DNA moves. This presents a problem because the electric field generates heat that must be dissipated or it will be absorbed by sample and the system. That can destroy the sample and affect the results. Moreover, electrophoresis is also used to refine material to be used in further testing. So instead of using a gel, a capillary can be used because they have a high surface area to volume ratios and that helps dissipate heat.

Making Larger Pieces into Smaller Pieces

Before separation and analysis, large DNA molecules are frequently divided into smaller fragments using a restriction enzyme (the fancy term is restriction endonuclease). In those instances where enzymes in the sample might affect the separation of molecules, these enzymes are removed before further analysis. After the DNA samples have been prepared, they are ready to be placed in the gel.  According to the literature, some laboratories may use two types of gel, agarose is used for long DNA molecules and polyacrylamide is used for short DNA molecules. Most of the time, the Gels are used (or run) in "slab" format while capillary electrophoresis is used for faster DNA sequencing.

Preparing the Gel

This is not an instruction sheet but an example of what might be entailed in preparing the gel, so please follow the manufacturer's instructions.  The Agar (agarose powder) is usually mixed with a buffering compound to the desired concentration and then heated in a microwave oven until the entire mixture is completely melted. Then at this point, ethidium bromide is added to the gel in a small concentration to help reveal details after the process of electrophoresis has been completed. After the liquid gel has cooled to about 60⁰ C, it is poured into a casting tray that has a comb at one end of the tray. It is then allowed to cool and solidify at 20⁰C.

After the gel has solidified, the technician carefully removes the comb making sure that the wells remain intact. Then the samples containing the DNA to be tested are pipeted into the sample wells. The gel is then covered with a buffering solution and then the tray is closed. Then the technician applies power. If the process is working, observers can probably see bubbles coming from the electrodes while the DNA is moving toward the positive electrode (anode). In direct current equipment, the anode (+) is usually colored red while the cathode is colored black (-).  During electrophoresis and after sufficient migration has occurred, DNA fragments can be seen more clearly by staining them with ethidium bromide and this is what is seen.

How Does it Work?

Gel electrophoresis can be used to identify the presence of DNA strands of a known length. In other words, particular DNA strands are much like a door key, they have a specific length, specific peaks and valleys, and other measurable attributes.

An enzyme is composed of complex proteins produced by living cells that catalyze specific biochemical reactions at body temperatures. A restriction enzyme can cleave a DNA molecule into fragments at specific sites inside it and this is why this type of enzyme is also called a restriction endonuclease. An endonuclease is an enzyme that breaks down a nucleotide chain into several shorter chains by cleaving the internal covalent bonds linking nucleotides.

A nucleotide is a compound that consists of a ribose or deoxyribose sugar and it is joined to a purine base or a pyrimidine base and to a phosphate group. These form the basic structure of nucleic acids such as RNA and DNA. A nucleoside is a compound (as guanosine or adenosine) that has a pyrimidine or a purine base combined with deoxyribose or ribose and is found especially in DNA or RNA.

DNA can be separated into individual strands using restriction enzymes that cut the DNA at specific, known locations. Then it is mixed with a dye or radioisotope that will allow its location in the gel to be identified. So the theorem is that individual strands can be separated from each other by using DNA electrophoresis. The process begins by injecting separated genetic material into wells that have been cut in the end of a slab of gel and then an electrical field is then applied to the gel.

Since DNA has a negative electric charge, it is attracted to the positive electrode but the gel resists the DNA as it moves; so the smaller pieces have an easier time moving through the pores in the gel and that is why they travel further. Since the technician knows density of the gel and electrical energy applied to the gel, the length of a DNA segment can be easily determined by the distance that it has traveled. Moreover, this means that the DNA segment can then be cut from the gel using a scalpel.

Two kinds of gels are used, Agarose and Polyacrylamide. Agarose is made from seaweed while Polyacrylamide is a synthetic polymer. Agarose is less dense than polyacrylamide and allows larger molecules to move through it. Conversely, polyacrylamide is more dense than agarose and allows only smaller molecules to move though it. So if the fragments are very short, a polyacrylamide gel is used. Otherwise, an agarose gel may be used. In other words, these gels act as a sieve that can be precisely calibrated. So if these fragments are very short, a polyacrylamide gel is used. Otherwise, an agarose gel may be used.

A method called pulsed-field gel electrophoresis must be used for very long segments. This process uses an electrical field that constantly undergoes changes in polarity. The process is similar to straightening a rope or a coil of garden hose. It does this by making small changes in the direction of current flow (alternating current) to keep very long strands correctly oriented as they move through the Agarose Gel.

Capillary Electrophoresis

Gel electrophoresis requires many steps from preparing the gel plates, making the gel, pouring it, waiting for it to set and then removing the comb. All of this takes time and the gels are toxic so this is why premanufactured gels are preferred. The problem is that someone has to carefully place each sample into its assigned well without contaminating the other samples.

Capillary electrophoresis removes the problem of working with gels because it takes advantage of the surface tension of a liquid which makes them assume the shape of having the least surface area. This also means that many processes can be automated, greatly reducing the probability of human error. Since a capillary is a very small tube, the most important benefit is that extremely minute samples can be used. This provides more material for retesting as the need might occur. An important aspect of capillary electrophoresis is that the results are displayed in a particular form format called an electropherogram. This format allows the DNA from different persons to be depicted as peaks or spikes in an easily understandable display. As a result, the signal strength of the DNA from each contributor will remain in constant proportion to each other with certain anomalous exceptions.

Tandem Repeats

A tandem repeat consists of an arrangement of two or more repeated nucleotides such that the repetitions are directly adjacent to each other. For an example, ‘AGCTAGCT’ represents a double repeat of the sequence ‘AGCT’ and ‘AGCTAGCTAGCT’ represents a triple repeat. When 10 to 60 nucleotides are repeated, the combination is called a ‘mini satellite’. Those combinations with fewer nucleotides such as ‘AGAG’ are called a ‘micro satellite’ or a short tandem repeat. However, when the number of repeats is variable or not known, they are called a ‘variable number tandem repeat’. The order of the nucleotides is important because these particular tandem repeats are useful in determining parentage through electrophoresis. This can be done by determining the percentage of the bands that match.

Summary

In electrophoresis, electric current repels negatively-charged DNA fragments through extremely small pores in the gel. In electron deficit theory, a positive anode attracts negatively charged DNA fragments through the microscopic pores in a gel.  So when the anode, cathode, and the DNA fragment are in equilibrium, no further movement will occur. Molecules with a stronger intrinsic electric charge will move faster than those with a weak charge.  In other words, molecular movement is governed by drag and so another defining characteristic of a molecule is its drag coefficient in a particular medium. So the larger and more asymmetrical molecules will move more slowly than slimmer and stream lined molecules. In protein electrophoresis, the proteins are often uniformly coated with an electric charge so that only their sizes and shapes are relevant.

I hope that I remember this correctly, Mel Brooks co-written and produced and directed a movie called ‘Men in Tights’. In this movie, a mole moved (I am referring to a pigmented mark or spot on the body and not the animal). Normally others would call this a continuity error but Mr. Brooks took advantage of this to make the motion picture humorous and enjoyable. Keep in mind that the movement of the mole is physical evidence but the mole could be a source of genetic evidence. In fact, DNA testing could reveal which actor or actors wore the mole.

When I began my paragraph on photographic examination, I wrote about continuity errors while referring to a pigmented mark or spot. These spots do not move and the body produces endorphins to ease the pain. As the bruise heals, the blood breaks down to pigments from red to blue to cyan and to yellow that are gradually absorbed into the bloodstream. Position and color change can be detected and recorded.  So the first detection method is blood testing for certain drugs and such as aspirin, warfarin, and other drugs that can cause that can cause extensive bruises. Bruises do not move but they may expand in the red to blue transition and most people know about this process. 

Edge detection is the graphic analysis of a photograph, an image, or a graphic design. It used in structural models to show the effect wind, explosions, earthquakes, or any other hazzard that can affect the integrity of a structure. Edge detection can also expose false accusations of spousal abuse. I was going to write more on edge detection but it is complex and the Internet has several articles on this subject. There is one axiom in edge detection. If nothing is missing and the edges don’t fit then the evidence is not wrong.

Back to the Past

Color television has been around for a long time. However, when television stations applied to the Federal Communications Commission (FCC) to transmit shows in color, the FCC required that these stations transmit their programs in compatible color. That meant that their transmitting signal could be received by the ‘Black and White’ television sets of that era. Those sets only had a single electron gun that modulated the intensity of the electron beam that struck a single phosphor giving a grey scale. In contrast, color televisions used three electron guns that modulated the intensity the output of three electron guns that struck three different phosphors giving red, green, and blue. That’s the simple explanation.

A pixel is a picture element and the RGB color pixel has three phosphors red, green, and blue. The three phosphors of an RGB pixel can be arranged as a horizontal line, a vertical line, a normal triangle, an inverted triangle, or a sequence of normal triangles alternating with inverted triangles.  The Hue, Intensity, and Saturation (HIS) model defines color formation. The hue is the based color, intensity is the freedom from dilution with black, and saturation is the freedom from dilution with white. For an example, dark red is created by a low intensity red without any amount of green and blue while pink is red diluted by equal amounts of green and blue.

Each phosphor is excited by an electron gun that controls the phosphor’s intensity. In an eight bit system, intensity is a number that ranges from zero to 255 where zero is the minimum intensity and 255 is the maximum intensity. If the intensities of three phosphors are equal, a gray scale shade is produced that ranges from black, through gray, to white. When the intensities of the three phosphors are not equal, a color scale shade is produced that ranges from black, through various colors, to white.

A three-color lookup table is partitioned to produce 256 entries of red, 256 entries of green, and 256 entries of blue giving 16,777,216 shades where 16,776,960 entries are colors and 256 entries are gray scale. This arrangement is easy to initialize because each partition is an exact duplicate of the other partitions. So the red partition defines the intensities of the red phosphors, the green partition defines the intensities of the green phosphors, and the blue partition defines the intensities of the blue phosphors. When the red, green, and blue intensities are equal, they define shade of gray ranging from black, to gray, to white.

Bringing Things to Light - Bruises

Years ago, the Jet Propulsion Laboratory (JPL) provided images of Io, a moon of Jupiter. The standard color lookup table with 256 entries didn’t reveal very much. So I used a three-color (RGB) lookup table with 16,777,216 entries and it revealed a volcanic explosion on the surface of Io. Today, most cameras use charge-coupled device technology and many include software that allows images to be stored on a computer for printing and analysis. It is important to remember is that the human perception of light and color is less linear and more logarithmic. So an image can be manipulated without altering its original rendering.  A library may have books that describe the process of analysis, and the enhancement of photographs.

A bruise (also called a contusion) is an injury to the skin and underlying tissues that produces bleeding under the skin. Most bruises are caused by a sudden blow that compresses or crushes the soft tissues, such as the skin and muscles. A severe blow can penetrate to the bone and cause a bone bruise. Bruises are initially treated by the application of cold compresses, ice packs, or endothermic chemical cold packs. The chemical cold packs are preferred because they are easy to store, they do not melt or drip, and do not injure tissues by freezing the affected area.  However, sometimes a bruise is caused by allergic reaction to aspirin or other substances. The abuse of aspirin, warfarin (coumadin) and the use of cosmetics and prosthetics can produce similar results and self-inflicted injuries or injuries inflicted by another person cannot be ignored. 

Bruises are often initially painful and the body produces endorphins to ease the pain. As a bruise heals, the blood breaks down to pigments from red to blue to cyan (greenish blue) and to yellow that are gradually absorbed into the bloodstream. Also the shape of a bruise can reveal the truth. Most bruises are not symmetrical but can have random shapes. The reason for this is that the capillaries are very small and like finger prints in identical twins, they are unique to each individual.  So it is important to use photographic means to record the location, symmetry, and color change of a bruise or other injury.

Contact DNA, Exchange DNA, and Splatter Back DNA

Contact DNA occurs when a person touches something. When one person touches another person, a DNA exchange occurs. The amount genetic material exchanged is highly variable because it is dependent upon force and duration. When a person touches an object, a small amount of their DNA is transferred to that object and any DNA that was on that object can be transferred to that person.  That's called an exchange.

Splatter Back occurs when somebody hits an object or another person. So the amount of splattered back DNA material transmitted from the assailant to the victim is greater than the DNA that is splattered back from the victim to the assailant. Another way of explaining splatter back is painting a painting a room with a roller. The faster the roller is moved or pressure is applied, more paint splattered back upon the painter and only a very small amount is spattered from the painter back onto the surface. This testing requires a very few cells but the problem is amplifying the sample without introducing or increasing contamination. So this kind of testing requires unique procedures because some samples may have an abundance of DNA that masks the presence of the DNA of interest.

A misnomer is the use of a wrong name or wrong designation. For an example, DNA finger printing is an analogy and a misnomer because it does not involve the physical aspect of finger printing and finger print examination. The difference is that DNA testing examines the genetic evidence and the fingerprint test examines the non genetic evidence.

Since DNA testing cannot distinguish genetically identical persons and each person has a unique set of finger prints, these become the discriminating factor for identifying individuals. However, finger prints can be etched to produce an exact copy but there is no genetic evidence transferred by the finger prints then they were faked. So proper testing requires both genetic and non-genetic evidence.

In the Beginning

Nature stores our genetic information in molecules of deoxyribonucleic acid (DNA). Each person's DNA is determined at conception when a sperm cell fertilizes an egg cell producing a zygote.  Non-identical twins (triplets etc) occur when separate egg cells are fertilized by separate sperm cells producing multiple unique zygotes.  DNA testing can differentiate between these persons.  When a zygote divides to form two or more separate copies of itself it creates genetically identical twins, triplets, quads, etc. that have the same DNA. Therefore, DNA testing cannot differentiate between these persons.  Each person has a unique set of figure prints and these figure prints can be used to identify a specific individual.

The DNA of a person is identical throughout a person's body. All human cells (except red blood cells) contain DNA. The DNA is determined at conception and does not change throughout the life of an individual.  All persons formed from a unique zygote have a unique DNA structure.  The same below is an illustration of a calculation of a DNA profile frequency and let’s look at the nomenclature (the designation of terms). A locus is the position of a particular gene or allele in a chromosome. Humans have 23 pairs of large linear nuclear chromosomes, where 22 pairs are called autosomes and one pair of sex chromosomes, for a total of 46 chromosomes.  Motherless DNA testing is often used to prove paternity (the father of a child).  This testing can produce a false positive result because the child, mother, and alleged father can have identical markers at the same locus (location) in the DNA molecule. That is why I do not approve of motherless DNA testing.

Human chromosomes are ordered (numbered) according to their size from the largest to the smallest. So the largest chromosome is designated by the numeral ‘1' and the smallest is designated by the number 22 and these are called the autosomes. The sex chromosomes are treated differently. Normally, they are paired to determine gender ‘XX’ for females and ‘XY’ for males. Moreover, the sex chromosomes have been found to exist as multiples greater than two such as 'XXY' or 'XYY' and other sequences.

Location is Everything

A chromosome has two telomeres ‘p’ and ‘q’ separated by a centromere. The letter ‘p’ is used to designate the shorter telomere and the letter ‘q’ designates the longer telomere. The bands of the chromosome are numbered outward from the centromere. If the telomeres are equal in length then 'pp' is used. (The reason that ‘p’ is used is that the letter ‘p’ occurs before the letter ‘q’ in the alphabet.) So the naming convention specifies the type of DNA, chromosome number, copy sequence, and order of discovery or registration. This is why D16S539 is the designation for DNA chromosome 16, ‘single copy sequence’ at locus 539.

In geometry ‘the locus of points’ refers to a set of points that have a particular position relative to another position called a fiduciary (a standard of reference) point. Often that particular position is called the origin and sometimes it is called the first point or the principal locus (location).  So an allele is an alternative form of a gene that may occur at a particular locus. In the example below, CSF1PO-10 and CSP1PO-11 are alleles that occur at a location. However, TPOX-8 does not have any variants or alleles because there are no alternative forms of this gene.

The sample table below shows that allele CSF1PO-1O was observed109 times in a sample of 432 and allele CSFP1PO-11 was observed 134 times in a sample of 432. So dividing the number of observations by the sample size gives the relative frequency (occurrences) of the two alleles of CSF1PO. So 109 divided by 432 is 0.25 (p) and 134 divided by 432 is 0.31. To compute the genotype frequency for the locus, the function ‘2pq’ is used. Therefore, 2x(0.25 x 0.31) is 0.155 and is rounded to 0.16 for this illustration.

TPOX-8 was observed 229 times in a sample of 432. The size of the data base was 442. So 229 divided by 432 is 0.53 giving ‘p’. To compute the genotype frequency for this locus, the function ‘pp’ is used. Therefore, (.53 x .53) is 0.28. So when there are multiple alleles, ‘2pq’ is used and when there only one occurrence, ‘pp’ is used.

The profile frequency is simply the product of all of the individual products for each allele or each single gene. So ((0.16) x (0.28) x (0.07) x (0.05)) is 0.00014. But what does this result show? Inverting the value reveals that there is only one person in 7,142 that has the same DNA profile. The problem is that the profile is not large enough and the product rule (pp and 2pq) can be inaccurate for some population mixtures.

This data was obtained from Forensic DNA Typing by John M. Bultler.  I have made an incomplete copy of data that is shown on page 502 of that book.  I have done this for the purpose of illustration on how this data is used to determine the product rule genotype frequency for a number of locations.  The Internet may have similar or better examples than what I have provided here.

Maternity Testing - Mitochondrial DNA

Mitochondrial DNA (mtDNA) is extracted from mitochondria that is located outside a cell’s nucleus and this explains one of several differences between mitochondrial DNA and nuclear DNA. The reason that mitochondrial DNA is important is that nuclear DNA degrades over time while mitochondrial DNA can be extracted from very old samples after nuclear the DNA has deteriorated. Moreover, mitochondrial DNA can be used to prove maternity since mtDNA is only inherited from the biological mother. This doesn’t mean that King Solomon would have been out of a job (I Kings 3:16-27).

Digital SLR Photography with Photoshop CS2 by Kevin Ames.
DNA Electrophoresis - Wikipedia
Forensic DNA Typing by John M. Bultler (IBSN-13: 978-0-12-147952-7)
Interpreting DNA Evidence by Ian W. Evett and Bruce S. Weir (ISBN 0-87893-155-4)
Mathematics: From the Birth of Numbers by Jan Gullberg
The Story of Numbers (Binomial Theorem) by John McLeish
Blood Stain Pattern Analysis from Wikipedia

Forensic Bioinformatics:  http://www.bioforensics.com

For detailed information on ABO blood testing on my website, please click here.

For additional information from Wikipedia on the ABO blood group system, please click here.

For additional information from Wikipedia on the Hh_antigen_system (Bombay Phenotype) - please click here.

For information on Sickle Cell Anemia - please click here.

For additional information on mutations and disorders - please click here.

For additional information from Wikipedia on Blood Stain Pattern Analysis - please click here.

For additional information from Wikipedia on gel electrophoresis - please click here.

For more information from the American Association of Blood Banks.

 Edward Steven Nunes

Index