• What is DNA?
• The Structure of DNA
• Variability in DNA
• Polymerase Chain Reaction (PCR)
• Visualization of the DNA

What is DNA?

DNA is short for deoxyribonucleic acid. DNA makes up the genetic "blueprint" which contains all the information necessary to make a living being.  DNA is found inside the nucleus of almost every cell in the human body, with the exception of red blood cells which do not have a nucleus at all.

DNA is wound into tight thread-like structures called chromosomes.  Humans have 46 chromosomes in total, half of which are inherited from the mother (in the egg), and half from the father (in the sperm).  44 of these chromosomes are called autosomes and the other two are sex chromosomes, known as the X and Y chromosomes.

We have two copies of each of the genes contained on the autosomes, one copy is inherited from our mother and the other from our father       

Refer to Diagram 1 in the  DNA Diagrams attachment- PDF file.                       

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DNA is a chemical molecule which is made up of a string of bead-like structures known as "bases" held together by a chemical backbone.   There are four types of bases in DNA - adenine, guanine, cytosine and thymine (A, G, C & T).  Two strands of DNA make up each chromosome, with the backbones forming a structure that is similar to the sides of a ladder.  The bases along the backbones pair up with weak chemical bonds to form the rungs of the ladder -  "A" pairs with "T", and "G" pairs with "C".  This means that the two strands are complementary to each other - if the sequence on one strand is AATCGT then the other strand will be TTAGCA.   This is called the DNA helix.

Refer to Diagram 2 in the   DNA Diagrams attachment - PDF file.  

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The order, or sequence, of the bases defines the information contained in the DNA.  A large proportion of our DNA is the same in all human beings, but there are some regions of DNA which show considerable variation between individuals in the population.  The variation that we are interested in is due to differences in the number of repeat sequences in a particular region of DNA.  We look at between eight and twelve separate variable regions (or loci) of DNA.  These  regions can be combined to generate a DNA profile, also known as a DNA "fingerprint".

These variable regions of interest are flanked by non-variable regions.  To examine the variable region, a common region on either side of the variable region can be targeted to pick out the area of interest from other DNA in the cell.  We are able to do this by using a technique called the Polymerase Chain Reaction (PCR) to target and reproduce thousands of copies of a particular variable region of DNA that we are interested in.  This then gives rise to DNA fragments that vary in length due to the variability in the number of repeated regions, as mentioned above.

Refer to Diagram 3 in the  DNA Diagrams attachment  - PDF file.

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The Polymerase Cahin Reaction (PCR) technique is used to amplify (copy) a small amount of DNA taken from a sample of blood, hair, tissue etc., to a much greater amount of DNA which can then be analysed.  Two short pieces of synthetic DNA called primers are specially designed to attach to a common non-variable region of DNA which flanks a variable region of DNA.  A mixture of chemicals including the primers, the individual bases and a copying enzyme (DNA polymerase) is added to a solution of template DNA.  The reaction is then subjected to a series of heating and cooling steps. 

In the first step the double-stranded DNA helix is separated (denatured) at 94ºC so that each back bone has unpaired bases along its length.   These bases inherently wish to re-pair with their complementary bases.  In the second step the reaction is cooled  and the short primer sequences attach to their target region, one at either end, but on opposing strands of the DNA template.  DNA polymerase adds the loose bases to the strand, generating a complementary strand of DNA based on the sequence of the template DNA.  This makes a double-stranded DNA molecule identical to the original.  The process is repeated, usually between 20-40 times, exponentially increasing the number of copies of the target region of DNA.

Refer to Diagram 4 in the  DNA Diagrams attachment - PDF file.   

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Once the target DNA is copied (amplified) it is visualized so that the DNA types can be determined.  At each of the 8-12 DNA regions that we look at, we will have two copies of the same gene.  As discussed above, we will receive one copy from our mother and the other from our father.  The two DNA types are called alleles.

The PCR process generates both alleles of the target region.  A person may have two copies of the same allele (homozygous) or two alleles of different lengths (heterozygous). 

The variablility in the regions of DNA that we use is in the length of the DNA strand that was amplified (due to variation in the number of repeated units of DNA sequence).  The DNA fragments are then separated on the basis of this difference in length by passing the DNA solution through a gel matrix by applying an electric current.  This process is known as gel electrophoresis.  The DNA is negatively charged and will move towards the positive electrode at a rate inversely proportional to its length, that is, the longer the fragment the slower it moves.

After electrophoresis is complete, the DNA is visualized in the gel matrix by staining with a silver-based compound.  This technique is very similar to the old method of developing photographs.  DNA fragments appear as dark bands, allowing the allele types to be assigned visually.  This is done by comparing them to a sample containing a number of known variants, known as an allelic ladder.  By amplifying a number of variable target regions a profile can be generated with two alleles at each region.  DNA profiles of different samples can then be compared for applications such as paternity testing, identification, sibship analysis and criminal investigations.

Refer to Diagram 5 in the  DNA Diagrams attachment  - PDF file.   

Note: The methods described above apply to the VIFM Molecular Biology laboratory at the current time.  They do not necessarily represent the methods that other laboratories are currently employing, nor do they represent all of the methods used by the VIFM .