Saturday, September 25, 2010

Polymarker (PM)

The PM portion of the PM plus DQA1TM  kit involves 5 genetic loci in addition to DQ alpha. (The manufacturer/ distributor's name has changed from Roche Molecular to PE Applied Biosystems).  These additional loci are named for historical reasons.  The 5 loci are LDLR, GYPA, HBGG, D7S8 and GC.  Each of these represents a distinct location or locus in the DNA.  The 5 non-DQ alpha loci have rather simple allelic variations compared to DQ alpha.  For example, there are only two LDLR alleles detected by the system, allele A and allele B.  The same is true for GYPA and D7S8 that each have A and B alleles that can be detected .  The loci, HBGG and GC each have A, B and C alleles, three variations each in other words.  Thus, reading PM typing strips is fairly simple at least on the surface.  Here are some examples:

 The manufacturer recommends a lower limit of input DNA for PM plus DQA1 typing.  The reason for this lower limit (2 nanograms, ng) is the possibility of missing alleles if the input DNA is too low.  Missing alleles (related terms are "allelic dropout" and differential amplification) is certainly possible in the author's opinion, particularly with low amounts of DNA or degraded DNA.  The phenomenon of failing to detect all alleles present was discussed in the context of DQ alpha in the original User's Guide  although the conditions under which this may occur were not precisely defined with respect to amount of input DNA and the condition of input DNA.  The potential for missing an allele of course increases if the control or S dot is absent or extremely weak, remembering that the C and S dots test whether a threshold has been reached at the PCR and later stages.  The following example illustrates how a DNA profile of one person might change to that of another due to failure to detect an allele. 

 Failure to detect alleles under certain circumstances is a theoretical probability and was actually demonstrated for DQ alpha in the original User's Guide.  The theory that addresses this is called, the "stochastic effect."  In addition to the stochastic effect, a PCR phenomenon called "differential amplification" may play a role when input DNA amounts are low, when input DNA is extensively degraded and possibly at other times. 
 PM plus DQA1 is frequently used on mixed DNA samples from two or more people.  The following example illustrates some of the ambiguity that can arise if interpretations are not cautious:

In the example above, since two of the loci (HBGG and GC) show three alleles, the sample was a mixture of at least two people.  The problem here is that any two people can be included as contributing to the mixture.  The typing strip is saturated, meaning every dot that can be showing is showing.  A poorly recognized limitation of the PM strip is that it is very easily saturated.  For example, two people of types AB/AA/AB/BB/BC (person 1) and AB/BB/AC/AA/AA (person 2) could, when their DNAs are mixed produce the pattern in the example.  In fact, there are almost limitless combinations of 2 types that could produce the pattern.  There are also many combinations of two people that would lead to a typing strip lacking one or two dots.  Finally, there are many mixtures that may mimic a single source of DNA.  For example:

 The profile in this example could have come from a single person whose profile was, AB/AB/AC/AB/AB.  Alternatively, two people of types AA/AB/AC/AA/BB and BB/AB/AA/BB/AA if mixed, could produce the profile.  There are many other possible combinations of people who, when their DNAs are mixed, could produce the profile.  In fact, the only individuals excluded are those possessing the HBGG, B allele and the GC, C allele assuming that the typing strip is reliably detecting all the alleles present.  Extreme caution should be used when there is a possibility of a DNA mixture.  It is arguable whether the system should be relied upon when there is an unresolved mixture.  The ease of saturation may lead to false inclusions.  False exclusions are also possible when the amount of input DNA is low, input DNA is degraded or the S dot is weak or absent.

Dot Hybridization

From the pattern of probes that the amplified DNA binds to, a potential DNA type, also called a genotype, can be inferred. 
DQ alpha typing strips look like this before any types are obtained:

 The invisible dot to the right of the number 1, has a DNA probe for the 1-allele (variation) for DQ alpha.  The invisible dot to the right of the 2 has a DNA probe for the 2-allele and so on.  The 1-allele itself has variations, the 1.1,1.2 and 1.3 subtypes, also called alleles.  Notice that the typing strip has no specific dot or probe for the 1.2 subtype.  Also, the typing strip can't distinguish between the 4.2 and 4.3 subtypes and there is a single dot for these.  It is quite possible that there exist DQ alpha alleles that would be undetected by the typing strip and alleles that may be further subtypes of the alleles that the strip does detect.
 Here are some examples of how the strips are read: 


This last example brings up an important issue with DQ alpha typing.  The 1.2 allele is actually the second most common allele in most populations.  This means there will be frequent situations where the 1.2 allele may be present but undetected as in the last example.  An obvious question is:  Why not just have a specific probe for the 1.2 allele?  The answer is that the typing strip already maximizes the probing of a relatively short stretch of DNA.  That is, the DQ alpha locus itself is only about 240 base pairs long.  The multiple probe typing strip was probably about the best that could be done in terms of detecting multiple alleles of this small locus in a single typing step.  
Historicall, DQ alpha was often the first PCR-based test that forensic labs used.  Actually, the DQ alpha system is quite different from the majority of PCR applications in the scientific community.  This will be explained in more detail below. 



DQA1 (also known as DQ alpha)

The PM plus DQA1TM (PE Applied Biosystems) typing kit targets six genetic loci.  All six are copied in the initial PCR.  The products from this reaction are then placed onto two separate typing strips.  One strip is for DQ alpha and the other types the remaining five loci. 
There are several steps in a DQ alpha PCR test: 
1.  DNA from 50 or more cells is extracted.  Notice that this test requires fewer cells that the RFLP test.  Sensitivity (the number of cells needed) is the main advantage of PCR tests.  However, the increased sensitivity also makes PCR tests more vulnerable to trace contaminants, DNA from unexpected sources, in other words. 
2.  The DNA from the sample is copied over and over resulting in amplification of the original target sequence.  The copying or amplification is accomplished in a machine specially designed for this purpose.  This machine is called a thermal cycler. 
3.  The amplified DNA is now treated with a variety of probes that are bound to a blot (see RFLP: Note: In RFLP, the target DNA is bound to the blot and the probe DNA is added.  For the DQ alpha dot blot, the probe DNAs are bound to a small blot strip and the target DNA is added).

Partial Profiles

Use of "partial profiles" is a newly emerging and fairly disturbing trend.  A partial profile is one in which not all of the loci targeted show up in the sample.  For example, if 13 loci were targeted, and only 9 could be reported, that would be termed, a partial profile.  Failure of all targeted loci to show up demonstrates a serious deficiency in the sample.  Normally, all human cells (except red blood cells and cells called "platelets") have all 13 loci.  Therefore, a partial profile represents the equivalent of less than a single human cell.  This presents some important problems: 
1.         A partial profile essentially proves that one is operating outside of well-characterized and recommended limits. 
2.         Contaminating DNA usually presents as a partial profile, although not always.  For this reason, the risk that the result is a contaminant is greater than for samples that present as full profiles.
3.         A partial profile is at risk of being incomplete and misleading.  The partial nature of it proves that DNA molecules have been missed.  There is no way of firmly determining what the complete profile would have been, except by seeking other samples that may present a full profile. 
Most forensic laboratories will try to obtain full profiles.  Unfortunately, in an important case, it may be tempting to use a partial profile, especially if that is all that one has.  However, such profiles should be viewed skeptically.  Over-interpretation of partial profiles can probably lead to serious mistakes.  Such mistakes could include false inclusions and false exclusions, alike.  It could be said that, compared to the first PCR-based tests introduced into the courts, use of partial profiles represents a decline in standards.  This is because those earlier tests, while less discriminating, had controls (known as "control dots") that helped prevent the use of partial profiles.  The earlier tests will be discussed below, primarily for historic reasons, but also because they do still appear on occasion.

Multiplex STR

One of the more commonly encountered STR test designs in forensic testing is called, Multiplex STR.  There are multiplex, PCR reagent kits sold by both:  PE Applied Biosystems and by  Promega.  Such systems combine three or more different PCRs in one reaction that targets distinct STR loci at the same time.  Three of the commonly used loci are called, CSF1PO, TPOX and THO1.  Again, the names of the loci have historical significance, but are of little importance as names. 
 Profiler Plus and CofilerTM (PE Applied Biosystems) combines 13 different STR loci.  PowerplexTM (Promega) uses the same 13 loci but the primers used are different. The Promega kit incorporates published primer sequences, a significant scientific advantage, since without the primer sequences, it is unclear which STRs at some loci are targeted.  A newer typing kit, IdentifilerTM (PE Applied Biosystems) incorporates the original 13 loci but adds 2 additional loci.  By design (meaning where the primers were placed on the DNA by the designers) multiplex STR loci have different, non-overlapping size ranges so that DNA fragments from the different loci will have different, non-overlapping size ranges.  Or, if the sizes overlap, they are tagged with differing dyes to help distinguish the 13 loci.  These test systems have boldly ambitious designs and should be considered fairly experimental, especially for samples whose quantity and/or quality is outside tested limits.   
There have been some discrepancies in profiles obtained with test kits from the two manufactures when the same samples were analyzed.  These discrepancies are not extremely common but are noticeable and fairly dramatic when they occur.  Any base within DNA can mutate (ie. change).  For example, an A base at a particular position can change to a G. Such mutations usually first appear in a sperm or egg cell.  Each mutation then appears throughout the body of the person who results from such a sperm or egg.   Discrepancies in test kit results are thought to be due to mutations in the sites that the primers bind.  These events are called, primer binding site mutations or PBS mutations.
Multiplex STRs are often combined with PCR for another locus called, amelogenin (pronunciation varies, but usually it is AM'-EEL-O-GEN-IN).  Amelogenin adds little to the discriminating power of the test.  Its purpose is to help distinguish male and female sources of DNA by detecting the X and Y chromosomes.  The amelogenin products have sizes that place them outside the size ranges of the other loci.  
Compared to PCR-based systems originally introduced, such as PM plus DQA1 (PE Applied Biosystems) multiplex STRs are technically more simple and direct at the allele detection stage.  On the other hand, multiplex STR are slightly more vulnerable to missing alleles.  There are two reasons for this.  1)Larger DNA fragments are degraded before smaller ones.  This is simply due to the fact that larger DNA molecules are bigger targets for degradative enzymes than smaller DNA molecules.  2)PCR itself favors (will produce more of) smaller DNA targets compared to larger ones that take more time to copy. The copying is done by a protein called an enzyme.  It can finish copying smaller DNA fragments more rapidly than larger ones.  
Both of these factors result in a tendency for small DNA fragments to be seen more readily than larger ones.  This is not an overwhelming tendency but certainly should be considered when amounts of input DNA are low, when DNA degradation is suspected, and particularly when a single small STR allele is weakly observed at a given STR locus.  
STRs are prone to an artifact called, "stutter bands" or "shadow bands."  These are thought to be due to the DNA repeats slipping out of register during the PCR process.  These are spurious PCR products that are usually one repeat length smaller than the main band.  The main problem that these pose is that it may be difficult to impossible to determine whether light intensity bands are due to stutter or due to presence of a mixture.  Although the stutter bands are predictably below (shorter than) the main band, the stutter bands do often align with common alleles. 

Most forensic laboratories are aware of stutter artifacts and many take extremely careful and appropriate countermeasures.  However stutter artifacts conceivably could play a role if inappropriate attempts are made to interpret minor components of a mixture.  
Some of the current STR detection/typing schemes use thin tubes called capillaries, instead of flat gels.  When a capillary is used, the results are often displayed  as tracings on a graph, instead of the image display shown above.  On such tracings, each main STR product will appear as a large peak while stutter bands appear as smaller  peaks (to the left).  The tracings are called, electropherograms (ELECTRO-FERO-GRAMS).  The tracing data should be accompanied by 

 numeric data that reveals:  the measured size of each PCR product, the intensity (peak height) and the estimated allele size.  The numeric data can be important in determining the quality of the results. 

 The two figures above show some alternative ways in which STR results/data are presented.  Basically the peaks represent tracings of bands that have come off the end of a gel, or may represent tracings of the gel itself, depending on the equipment used.  Larger DNA fragments are on the right and smaller ones on the left.  There are recommended standards, called thresholds for how high or low the peaks may be.
All technology has limitations.  For multiplex PCRs, the most serious limitations are in the areas of samples that are minimal, degraded, mixed, over-interpreted, contaminated or even potential combinations of these.  Some current practices lack support by the established literature.  Over-interpretation can also occur when there are partial profiles.[1]  The scientific system recognizes the human tendency toward over-interpretation and offers the countermeasures:  independent review, independent verification, scientific controls and demonstrations of reproducibility.  These reviews and controls are considered integral parts of the scientific process.
PCR-based testing is potentially useful since it is currently the only quick method of amplifying really minuscule amounts of DNA.  However, it is important to recognize that PCR based methods are exquisitely sensitive to contamination and need to be interpreted with extreme caution.  Match probabilities generated with some STR typing systems may involve extreme numbers perhaps giving the impression of an infallible result.  Scientific rigor often requires that extreme numbers be placed in a context that considers all aspects of testing including laboratory error rates and technical limitations.

What is the complement of a DNA sequence?

  This might be more information than you would like, but to really understand PCR primers, try to walk through this: 
The complement of a DNA sequence is the sequence written backwards exchanging all A's for T's, all T's for A's, all G's for C's and all C's for G's.  For example, the complement of the sequence, AGTA is TACT.  An easy way to get the complement of a DNA sequence is to write another line below the original sequence remembering that A replaces T and G replaces C.  Then read the lower line backwards: 
So, for the sequence:
 write the complementary line below it giving:
 Then, just read the lower line backwards (from right to left) giving the complement: 
In practical words, the upstream (left) primer can be a direct reading of the target sequence while the downstream primer (right) must be the complement of the directly read sequence.  
If the above is confusing, it may suffice to think of the primers as  two arrows that point at one another with the STR located between them.  This is how the PCR targets the locus and the STR.
In practice, PCR primers are usually at least 17 bases in length.  The point here is that to use PCR to target an STR, the primers recognize constant, conserved sequences that flank the actual STR.  This means that the actual length of the target sequence depends on where the primers are placed in the flanking sequence.  For example, the Promega and PE, Applied Biosystems test kits use mostly different primers.  For example, the upstream primer could be designed to recognize DNA 100 bases upstream of the sequence shown.  Similarly, the downstream primer could be designed to recognize DNA further downstream.  Such placement of the primers by design, further upstream and downstream, would make all alleles (variations) of the STR appear to be larger than if the primers are placed by design close to the STR itself.  Wherever the primers are placed, that defines the region we will examine.  That region will then vary among individuals due to changes in the STR itself as explained above for the simple STR based on the repeating AT motif.

After PCR is used to provide many copies of a given person's STR, the products (copies) are separated according to size on an electrophoretic gel (see RFLP above for more details about gels).  The gel can be flat, as for RFLP, or it can be in a round tube, called a capillary with a detector at the end of it.  Typical flat gel STR results look like this:
 The black bars are called bands.  Each band is made up of many identical-size DNA molecules that were produced by PCR.  The gel separates smaller bands (DNA molecules) from larger ones.  The bands near the lower end of the gel are smaller (ie. the DNA fragments are shorter in length)  than those near the top.  For example, looking at the reference ladder, the first band near the lower end of the gel is the smallest STR.  For simplicity, let's say this smallest band contains a single repeat such as CATG, flanked by other DNA that the primers actually recognize in everyone's DNA.  The next higher band in the ladder would then contain 2 repeats, CATGCATG; the next 3 repeats and so on.  By comparing the positions of bands in the unknown samples with the reference ladder, the allele sizes are deduced.  In this example, Sample A had bands at the 2-repeat position and the 5-repeat position. Common terminology would call this sample a 2,5 type.  Sample B would be called,  2,4.  For a single person, each locus normally has two alleles and these can be different (heterozygous) or the same (homozygous). 


This will be presented in some detail because STRs are important in current, forensic DNA testing.  The abbreviation, STR stands for Short Tandem Repeat.  STRs are the type of DNA used in most of the currently popular forensic DNA tests.  STR is a generic term that describes any short, repeating DNA sequence.  For example, the DNA sequence ATATATATATAT is an STR that has a repeating motif consisting of two bases, A and T.  It turns out that our DNA has a variety of STRs scattered among DNA sequences that encode cellular functions.  For reasons that are not entirely understood, people vary from one another in the number of repeats they have, at least for some STR loci.  For example, person #1 may have ATATAT at a particular locus while person #2 may have ATATATATATAT.  Thus, STRs are often variable (polymorphic) and these variations are used to try and distinguish people.  The term, STR doesn't necessarily imply PCR.  PCR is one of many methods that might be used to help analyze STRs.  STRs have also been analyzed by DNA sequencing for example.  To understand PCR-assisted STR typing, it is useful to briefly consider how such PCRs are designed.
Suppose that laboratory data revealed the following DNA sequence:
The STR is underlined and consists of the sequence, GATA repeated 7 times.  The dashes at the beginning and end of the overall sequence shown indicate that there is more sequence available both upstream and downstream of the region shown.  Remember, DNA is relatively very long and linear and we are just going to look at a small region of it. 
Now, let's say we want to design a PCR to examine this same locus in other people.  To design the PCR, we need two primers, short synthetic DNA molecules that recognize the region.  One primer might be, ATGCTAGTA (Italics, in the above sequence) a sequence that would recognize the DNA flanking the left side of the STR.  The second primer might be, AAAAAAAATTTTTT.  This is called the downstream primer and it might be difficult to recognize in the sequence.  The reason it is difficult to recognize at first is that it is the complement of the sequence, AAAAAAAATTTTTT (italics, on the right in the longer sequence above).  See "General Considerations", for a more detailed discussion.

PCR Contamination

            It is worth considering contamination early in this discussion since this is a well-recognized limitation.  Unfortunately, the importance contamination in PCR is often underestimated.  PCR copies DNA efficiently if the initial DNA is in good condition.  A single DNA entity (molecule) can become millions or billions of DNA molecules in about three hours.  The PCR process is sometimes compared to a Xerox machine since many copies are made.  While initially, this is a useful comparison, it doesn't communicate the true, chain-reaction nature of PCR.  In PCR, the original DNA is copied, then the copies are copied, those copies are copied and so on.  This results in dramatic increases in the amount of DNA that couldn't be easily accomplished in the Xeroxing analogy.  The PCR process deserves its classification as a "chain-reaction" because it has much in common with other chain reactions such as avalanches. 
            PCR is also very similar to what happens when a clinical infection occurs.  Clinicians have known for many years that a single germ (bacterial cell or virus) contaminating a wound can produce a massive infection.  Similarly, a DNA molecule can contaminate (infect) a PCR and become a significant problem.  The ability of small amounts of DNA to produce false and misleading results is well-known and well-documented within the research community, where the technology originated.  Anyone who has caught a cold from an unknown source, or who has a pollen allergy should have some sense of how easily PCRs are contaminated.  Actually, it is probably easier to contaminate a PCR than to catch a cold since unlike our bodies, PCRs lack immune systems.  The only protection PCRs have is the technique of the analyst, use of control samples to monitor contaminants and careful interpretation. 
            Prevention of false results involves the use of carefully applied controls and techniques.  As described later, such controls and techniques can rarely guarantee that contamination hasn't influenced the results.  In forensic DNA testing, some of the scientifically worst-case scenarios can be prevented by keeping DNA samples from known individuals well out of range of other items of evidence at all stages.  Most forensic DNA laboratories perform negative controls, blank samples that will often detect contaminants in the laboratory.  The blanks detect contaminants by showing partial or full DNA profiles representing the contaminants.  Alternatively, the blank may show no profile, consistent with, but not proving that contamination didn't occur.  Unfortunately, a few forensic DNA laboratories omit their controls.  A few favor the controls by using special equipment on them, or by not carrying them through the entire procedure.  Such practices are hazardous, especially when an important evidentiary sample has a low amount of DNA, degraded DNA, or otherwise presents as a minimal or partial (see below) sample.  In short, while PCR is a useful research tool, all applications require extreme care and vigilance.  


PCR is an abbreviation for "polymerase chain reaction."   (POLL'-IM-ER-ACE).  This term applies to a wide variety of different DNA tests that differ in reliability and effectiveness.   Reliabilities of each kind of PCR test need independent verification.  PCR itself doesn't accomplish DNA typing, it only increases the amount of DNA available for typing. 
PCR uses constant regions of DNA sequence to prime the copying of variable regions of DNA sequence.

PCR typically uses two short pieces of known DNA called primers (small arrows below).  These serve as starting points for the copying of a region of DNA.

Many forensic laboratories use commercial supplied DNA testing kits that contain key components for certain PCR-based tests.  PM plus DQA1TM, Profiler PlusTM and CofilerTM and IdentifilerTM are all test kits commercially supplied by PE Applied Biosystems.  PowerPlexTM is another test kit with variations supplied by Promega.  PowerPlex kits have published primers, an advantage if the precise DNA targeted is to be recorded for posterity or studied for research.  As of 2005, Profiler PlusTM and CofilerTM and PowerPlexTM are probably the most commonly used test kits in US forensic laboratories.  


 RFLP has been almost entirely replaced by PCR-based testing.  The following description of RFLP is included here primarily for historic reasons (more current formats see below).
 RFLP DNA testing has four  basic steps:
1.  The DNA from crime-scene evidence or from a reference sample is cut with something called a restriction enzyme.  The restriction enzyme recognizes a particular short sequence such as AATT  that occurs many times in a given cell's DNA.  One enzyme commonly used is called Hae III (pronounced: Hay Three) but the choice of enzyme varies.   For RFLP to work, the analyst needs thousands of cells.  If thousands of cells are present from a single individual, they will all be cut in same place along their DNA by the enzyme because each cells DNA is identical to every other cell of that person.
2.  The cut DNA pieces are now sorted  according to size by a device called a gel.  The DNA is placed at one end of a slab of gelatin and it is drawn through the gel by an electric current.  The gel acts like a sieve allowing small DNA fragments to move more rapidly than larger ones.  
3.  After the gel has separated the DNA pieces according to size, a blot or replica of the gel is made to trap the DNA in the positions that they end up in, with small DNA fragments near one end of the blot and large ones near the other end.  The blot is now treated with a piece of DNA called a probe.  The probe is simply a piece of DNA that binds to the DNA on the blot in the position were a similar sequence (the target sequence) is located. 
4.  The size or sizes of the target DNA fragments recognized by the probe are measured.  Using the same probe and enzyme,  the test lab will perform these same steps for many people.  These sizes and how they distribute among large groups of people form a database.  From the database a rough idea of how common a given DNA size measured by a given probe is found.  The commonness of a given size of DNA fragment is called a population frequency.

The restriction enzyme cuts the DNA into thousands of fragments of nearly all possible sizes.  The sample is then electrophoretically separated.  The DNA at this point is invisible in the gel unless the DNA is stained with a dye.  A replica of the gel's DNA is made on something called a blot (also called a Southern blot) or membrane.  The blot is then probed (mixed with) a special preparation of DNA that recognizes a specific DNA sequence or locus.  Often, the probe is a radioactively labeled DNA sequence (represented by * labeled object in the figure above).  Excess probe is washed off the blot, then the blot is laid onto X-ray film.  Development reveals bands indicating the sizes of the alleles for the locus within each sample.  The film is now called an "autorad."  The band sizes are measured by comparing them with a "ladder" of known DNA sizes that is run next to the sample.  A match may be declared if two samples have RFLP band sizes that are all within 5% of one another in size.  
For RFLP analysis to be reliable, all complex steps of the analysis must be carefully controlled.  Databases must be large meaning they include many people; they must be representative of the potential test subjects.  Because of the complexities of populations, databases must be interpreted with extreme care.  For example, DNA fragment sizes rare in one population may be very common in other populations.  Further, sub-populations or populations within populations must be considered.


There have been two main types of forensic DNA testing.  They are often called, RFLP and PCR based testing, although these terms are not very descriptive.  Generally, RFLP testing requires larger amounts of DNA and the DNA must be undegraded.  Crime-scene evidence that is old or that is present in small amounts is often unsuitable for RFLP testing.  Warm moist conditions may accelerate DNA degradation rendering it unsuitable for RFLP in a relatively short period of time. 
PCR-based testing often requires less DNA than RFLP testing and the DNA may be partially degraded, more so than is the case with RFLP.  However, PCR still has sample size and degradation limitations that sometimes may be under-appreciated.  PCR-based tests are also extremely sensitive to contaminating DNA at the crime scene and within the test laboratory.  During PCR, contaminants may be amplified up to a billion times their original concentration.  Contamination can influence PCR results, particularly in the absence of proper handling techniques and proper controls for contamination.
PCR is less direct and somewhat more prone to error than RFLP.  However, PCR has tended to replace RFLP in forensic testing primarily because PCR based tests are faster and more sensitive.  

What is a Chromosome?

When a cell is getting ready to divide creating two daughter cells, it packs its DNA into bundles called chromosomes.  Chromosomes are just bundles of DNA.  For humans, there are consistently 23 pairs of chromosomes, each with a consistent size and shape.  Chromosomes are numbered.  Chromosome number 1 is the largest chromosome; chromosome number 2 a little smaller and so on.  Among the 23 pairs of chromosomes there is a pair called the sex chromosomes.  This is something of a misnomer, since there are many functions on the "sex" chromosomes that have nothing to do with sex.  In females, the sex-chromosome pair consists of two similar size chromosomes called X chromosomes.   Males have one X and one small Y chromosome.

Unless it has been purified, our DNA is actually not a loosely tangled string as illustrated but rather is well organized and packaged into what are called chromosomes.  A chromosome is a tightly folded bundle of DNA.  Chromosomes are most visible when cells divide.  In a microscope, chromosomes look something like this without the numbers and letters:

The illustration shows a pair of chromosomes named chromosome number 4, one pair among 23 pairs of chromosomes.  The illustration also shows the position of a locus that happens to be called "GYPA."   In this example, the chromosome on the left has the variation called the B allele while the chromosome on the right has the variation called the A allele. 
What are alleles?
Alleles (ALL-EELS') are just variations at a particular site on a chromosome.  Since each chromosome has a similar chromosome partner (except for males with their X and Y chromosomes) each locus is duplicated.  Loci can vary a bit.  If a person has two identical versions of the locus, they are said to be homozygous (HOMO-Z-EYE'-GUS).  If there is a difference, they are said to be heterozygous (HETERO-Z-EYE'-GUS).


DNA is material that governs inheritance of eye color, hair color, stature, bone density and many other human and animal traits.  DNA is a long, but narrow string-like object.  A one foot long string or strand of DNA is normally packed into a space roughly equal to a cube 1/millionth of an inch on a side.  This is possible only because DNA is a very thin string.
Our body's cells each contain a complete sample of our DNA.  One cell is roughly equal in size to the cube described in the previous paragraph.  There are muscle cells, brain cells, liver cells, blood cells, sperm cells and others.  Basically, every part of the body is made up of these tiny cells and each contains a sample or complement of DNA identical to that of every other cell within a given person.  There are a few exceptions.  For example, our red blood cells lack DNA.  Blood itself can be typed because of the DNA contained in our white blood cells.  
Not only does the human body rely on DNA but so do most living things including plants, animals and bacteria.  
A strand of DNA is made up of tiny building-blocks.  There are only four, different basic building-blocks.  Scientists usually refer to these using four letters, A,  T,  G,  and C.  These four letters are short nicknames for more complicated building-block chemical names, but actually the letters (A,T, G and C) are used much more commonly than the chemical names so the latter will not be mentioned here.  Another term for DNA's building blocks is the term, "bases."  A, T, G and C are bases. 
For example, to refer to a particular piece of DNA, we might write:  AATTGCCTTTTAAAAA.  This is a perfectly acceptable way of describing a piece of DNA. Someone with a machine called a DNA synthesizer could actually synthesize the same piece of DNA from the information AATTGCCTTTTAAAAA alone.  
The sequence of bases (letters) can code for many properties of the body's cells.  The cells can read this code.  Some DNA sequences encode important information for the cell.  Such DNA is called, not surprisingly, "coding DNA."  Our cells also contain much DNA that doesn't encode anything that we know about.  If the DNA doesn't encode anything, it is called non-coding DNA or sometimes, "junk DNA."[1]  
The DNA code, or genetic code as it is called, is passed through the sperm and egg to the offspring.  A single sperm cell contains about three billion bases consisting of A, T, G and C that follow each other in a well defined sequence along the strand of DNA.  Each egg cell also contains three billion bases arranged in a well-defined sequence very similar, but not identical to the sperm. 
Both coding and non-coding DNAs may vary from one individual to another.  These DNA variations can be used to identify people or at least distinguish one person from another. 
What is a Locus?
A locus (with a hard "c", LOW-KUS)  is simply a location in the DNA.  The plural of locus is, loci ( with a soft "c", pronounced LOW-S-EYE).  Again, the DNA is a long string like object as illustrated below.  A locus is simply a location in the DNA.  Such locations, or loci, reside at specific places on chromosomes.

Introduction to dna test procedure

 The explanation of DNA testing that follows is intended as an introduction to the subject for those who may have limited backgrounds in biological science.  While basically accurate, this explanation involves liberal use of illustration and, in some cases, over-simplification.  Although intended to be informative, this is brief and incomplete explanation of a complex subject.  The author suggests consulting the scientific literature for more rigorous details and alternative views. 

Paternity Testing Procedure

To start a paternity test, simply call 1-800-613-5768 or use the online order form. Our client service team will take your information and arrange for convenient sample appointments; we can schedule separate appointments for different parties if required.
At the Collection Site
All test participants are asked to present government-issued identification (such as a Driver’s License) and to complete a Client Identification and Consent Form. The legal guardian must sign this form for test participants who are minors. The sample collector takes fingerprints and photographs of all test participants.
Buccal Swab Collection
Samples for a DNA test, such as a paternity test, are routinely collected using the painless and simple buccal swab—similar to a cotton-tipped swab, but made of the special material Dacron®. The swab is rubbed against the inside cheek of the test participant, and loose cheek cells adhere to the swab. Unlike regular cotton swabs, Dacron® provides a consistent surface for sample collection and DNA extraction.
DNA Test in the Laboratory
The sample collector sends the swabs to our laboratory in a sealed, tamper-evident package. When samples arrive in our laboratory, our staff checks to see if the package seal is intact. Each sample is recorded into our database. When all the samples for a paternity test case have arrived, we start the DNA test, which follows the following five steps:
  • Samples from each person are divided in two for Dual Process™. From this point on, two independent laboratory teams take the samples through the DNA testing process.
  • DNA is extracted from the buccal swabs and purified.
  • The extracted DNA is added to a special chemical mix for the Polymerase Chain Reaction (PCR), a process that targets 16 specific locations in the DNA (called loci) and makes billions of copies of each location.
  • The products of PCR are analyzed to create a DNA profile, a genetic equivalent of a fingerprint for each tested party.
  • The child’s DNA profile is compared with the alleged father’s, and statistical analysis is performed to determine the probability of paternity. A 0% probability of paternity represents an exclusion (the alleged father is not the biological father) and at least a 99.99% probability represents an inclusion in a standard trio (the alleged father is the biological father).
Paternity Test Report
The paternity test results are ready within 3 working days (5 days for prenatal tests). We release the test results immediately, as long as the testing fees are fully paid. Results are sent via first-class mail; we also will send results through fax or courier upon request. Please call 1-800-613-5768 for instructions on requesting special delivery of results.

Signs and tests

Muscle wasting begins in the legs and pelvis, then progresses to the muscles of the shoulders and neck, followed by loss of arm muscles and respiratory muscles. Calf muscle enlargement (pseudohypertrophy) is quite obvious. Cardiomyopathy(DCM) is common, but the development of congestive heart failure or arrhythmias (irregular heartbeats) is only occasional.

    * A positive Gowers' sign reflects the more severe impairment of the lower extremities muscles. The child helps himself to get up with upper extremities: first by rising to stand on his arms and knees, and then "walking" his hands up his legs to stand upright.
    * Affected children usually tire more easily and have less overall strength than their peers.
    * Creatine kinase (CPK-MM) levels in the bloodstream are extremely high.
    * An electromyography (EMG) shows that weakness is caused by destruction of muscle tissue rather than by damage to nerves.
    * Genetic testing can reveal genetic errors in the Xp21 gene.
    * A muscle biopsy (immunohistochemistry or immunoblotting) or genetic test (blood test) confirms the absence of dystrophin, although improvements in genetic testing often make this unnecessary.

Mechanical ventilatory/Respiration Assistance

Modern "volume ventilators/respirators," which deliver an adjustable volume (amount) of air to the person with each breath, are valuable in the treatment of people with muscular dystrophy related respiratory problems. Ventilator treatment can begin in the mid to late teens when the respiratory muscles can begin to collapse. However there are people with the disease in their 20's who have no need for a ventilator.

If the vital capacity has dropped below 40 percent of normal, a volume ventilator/respirator may be used during sleeping hours, a time when the person is most likely to be under ventilating ("hypoventilating"). Hypoventilation during sleep is determined by a thorough history of sleep disorder with an oximetry study and a capillary blood gas (See Pulmonary Function Testing). The ventilator may require an endotracheal or tracheotomy tube through which air is directly delivered, however, for some people delivery through a face mask is sufficient.

If the vital capacity continues to decline to less than 30 percent of normal, a volume ventilator/respirator may also be needed during the day for more assistance. The person gradually will increase the amount of time using the ventilator/respirator during the day as needed. A tracheotomy tube may be used in the daytime and during sleep, however, delivery through a face mask may be sufficient. The machine can easily fit on a ventilator tray on the bottom or back of a power wheelchair with an external battery for portability.

Treatment for Duchenne muscular dystrophy

There is no known cure for Duchenne muscular dystrophy, although recent stem-cell research is showing promising vectors that may replace damaged muscle tissue. Treatment is generally aimed at controlling the onset of symptoms to maximize the quality of life, and include the following:

    * Corticosteroids such as prednisolone and deflazacort increase energy and strength and defer severity of some symptoms.
    * Randomised control trials have shown that beta2-agonists increase muscle strength but do not modify disease progression. Follow-up time for most RCTs on beta2-agonists is only around 12 months and hence results cannot be extrapolated beyond that time frame.[citation needed]
    * Mild, non-jarring physical activity such as swimming is encouraged. Inactivity (such as bed rest) can worsen the muscle disease.
    * Physical therapy is helpful to maintain muscle strength, flexibility, and function.
    * Orthopedic appliances (such as braces and wheelchairs) may improve mobility and the ability for self-care. Form-fitting removable leg braces that hold the ankle in place during sleep can defer the onset of contractures.
    * Appropriate respiratory support as the disease progresses is important

Symptoms of Duchenne muscular dystrophy

The main symptom of Duchenne muscular dystrophy, a progressive neuromuscular disorder, is muscle weakness associated with muscle wasting with the voluntary muscles[citation needed]  being first affected, especially affecting the muscles of the hips, pelvic area, thighs, shoulders, and calf muscles. Muscle weakness also occurs in the arms, neck, and other areas, but not as early as in the lower half of the body. Calves are often enlarged. Symptoms usually appear before age 6 and may appear as early as infancy. The other physical symptoms are:

    * Awkward manner of walking, stepping, or running. (patients tend to walk on their forefeet, because of an increased calf tonus. Also, toe walking is a compensatory adaptation to knee extensor weakness.)
    * Frequent falls
    * Fatigue
    * Difficulty with motor skills (running, hopping, jumping)
    * Increased Lumbar lordosis, leading to shortening of the hip-flexor muscles. This has an effect on overall posture and a manner of walking, stepping, or running.
    * Muscle contractures of achilles tendon and hamstrings impair functionality because the muscle fibers shorten and fibrosis occurs in connective tissue
    * Progressive difficulty walking
    * Muscle fibre deformities
    * Pseudohypertrophy(enlarging) of tongue and calf muscles. The enlarged muscle tissue is eventually replaced by fat and connective tissue, hence the term pseudohypertrophy.
    * Higher risk of behavior and learning difficulties, due to adjustment problems and muscular fatigue.
    * Eventual loss of ability to walk (usually by the age of 12)
    * Skeletal deformities (including scoliosis in some cases)

Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe recessive X-linked form of muscular dystrophy  characterized by rapid progression of muscle degeneration, eventually leading to loss of ambulation and death. This affliction affects one in 3500 males, making it the most prevalent of muscular dystrophies. In general, only males are afflicted, though females can be carriers. Females may be afflicted if the father is afflicted and the mother is also a carrier/ affected. The disorder is caused by a mutation in the dystrophin gene, located in humans on the X chromosome (Xp21). The dystrophin gene codes for the protein dystrophin, an important structural component within muscle tissue. Dystrophin provides structural stability to the dystroglycan complex (DGC), located on the cell membrane.

Symptoms usually appear in male children before age 5 and may be visible in early infancy. Progressive proximal muscle weakness of the legs and pelvis associated with a loss of muscle mass is observed first. Eventually this weakness spreads to the arms, neck, and other areas. Early signs may include pseudohypertrophy (enlargement of calf and deltoid muscles), low endurance, and difficulties in standing unaided or inability to ascend staircases. As the condition progresses, muscle tissue experiences wasting and is eventually replaced by fat and fibrotic tissue (fibrosis). By age 10, braces may be required to aid in walking but most patients are wheelchair dependent by age 12. Later symptoms may include abnormal bone development that lead to skeletal deformities, including curvature of the spine. Due to progressive deterioration of muscle, loss of movement occurs eventually leading to paralysis. Intellectual impairment may or may not be present but if present, does not progressively worsen as the child ages. The average life expectancy for patients afflicted with DMD varies from late teens to early to mid 20s. There have been reports of a few DMD patients surviving to the age of 40, but this is extremely rare.[citation needed]