RFLP EXPLAINED IN EASY TERMS

 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.

FORENSIC DNA TESTING

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 EXPLAINED IN EASY TERMS

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.