Is it Possible that DNA Genetic Material itself to fool the Definite Determination? Part VI - a recap and expansion

Real Fathers may escape responsibility of fathering; Lawyers shall stand on their toes on floor to question the protocols used by laboratories, to determine a real father. 

In the last episode we saw that there existed many hurdles in using DNA typing for any forensic investigation. The amount of sample that have to be collected, and adequate amount of DNA that can be extracted for the analytical methods in use during nineteen eighties and into the early nineteen nineties were of very challenging.  Extraction of DNA became sensationally possible virtually from any tiny material and analytical procedures as well improved day by day, that made the required amount DNA for a procedure also, very minimal with even more sensationally. Having solved these, the second hurdle faced by scientists was target sequences within the DNA extracted. Originally they were targeting coding sequences of real genes which is only represents a minimal 2% of entire Nucleotide base pairs of 3.3 billion for a human and other mammals and vertebrates bit higher 3% to 20%. This gene codon targets gave enough trouble to laboratories since mutations in DNA, due to environmental pressure and impacts. Human usually lives with about 400 odd Mutations within DNA without any apparent defects.  It is best to remember loci length are measured in base pairs (bp) and we saw that using intron became ethical, the obvious reasons for that the noncoding intronic codons are very conservative do not easily undergo mutation during the replication of DNA, higher the animal in evolution tree, higher the percentage of noncoding introns within the DNA helix molecule. Repetitive sequences between two loci very distinctive, and numerous making it easy specific for targets for forensic and anthropological studies.  

Once the targets have been located within the extracted DNA, fragmenting is a relatively easy process by the use of specific enzymes as it acts only on targets.  Mitochondrial DNA does not contain Non-coding Introns. Anthropological studies and Forensic science markedly improved mainly by ability to recover DNA that can be processed from frozen and dormant biological as well as from inert materials, and main the objective of polymerase chain reaction (PCR) is to produce enough DNA template and huge number of copies of each to work with for further analysis and typing from recovered sample.  

Polymerase Chain Reaction (PCR)

 A revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. The first and most commonly used of these enzymes is Taq DNA polymerase  (from bacteria Thermis aquaticus), whereas Pfu DNA polymerase (from Bacteria  Pyrococcus furiosus) is used widely because of its higher accuracy when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR because they can generate new strands of DNA using a DNA template and primers - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands and because essentally they are heat resistant,  Extremely variable regions, characteristic of non-coding DNA are used. Genetically different individuals produce different profiles. The closer the genetic relationship between individuals the more similar their profiles

1.  The DNA is released from a cell:  The cells of an uncontaminated biological sample (blood, semen, hair root, cheek cell) are broken open and the DNA is released and separated. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction within in a tube for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

2.            There are 3 steps initially

A.    The cycling reactions :

The three initial steps in a PCR that are repeated for 30 or 40 cycles. This is done on an automated machine which can heat and cool the tubes with the reaction mixture in a very short time. I. Denaturation at 94°C: during the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle) II. Annealing at 54°C: the primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. III. Extension at 72°C: This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of fragment. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

B.     Is there a gene copied during PCR and is it the right size?

Before the PCR product is used in further applications, it has to be checked if

a.      There is a product formed. Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template

b.     The product is of the right size, It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene.

c.      Only one band is formed. As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel.

Electrophoresis Verification,

The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1: PCR fragment is approximately 1850 bases long. Lane 2 and 4: the fragments are approximately 800 bases long. Lane 3: no product is formed, so the PCR failed. Lane 5: multiple bands are formed because one of the primers fits on different places. a constrain of PCR method. 

Having completed enough DNA recovering process, actual analysis commenced as described last episode. 

3.        Digestion: Special enzymes are used to cut the DNA at specific points and produce a set of fragments of varying lengths. These enzymes are called restriction enzymes. The DNA sections that are cut are called restriction fragments. Certain enzymes will cut the DNA at specific bases (T, G, A, and C). Each restrictive fragment will be specific for that particular person. The bases as well as the distance between the bases (non-coding DNA) are characteristics of that individual organism (person).

4.              Separation: The fragment mixture is placed in a block of gel and separated by gel electrophoresis on the basis of size. The fragments are exposed to an electrical charge. This causes the fragments to move. The smaller the fragment the further it travels. The bands of small fragments are separated from the bands of the large fragments. After other treatments, a photographic copy of the pattern of DNA is compiled. Using trade mark method the positions of the DNA fragments on X-ray film appear as dark bands. The positions of these bands are the genetic profile of the individual and the more numerous the bands the more reliable the ‘fingerprint’. The bands are produced and vary in thickness depending on how many DNA fragments are present in a certain length of DNA. 

5.  Pattern comparison: The DNA fingerprint can be used to be compared to other DNA fingerprints. The pattern is unique to an individual. In this way comparisons of known DNA to unknown DNA can be made to determine if the unknown DNA can be identified.  

Applications of DNA Profiling

1. Crime: Forensic medicine uses medical evidence in legal disputes. DNA profiling is used in forensic medicine. Material such as hair, semen, blood, and saliva which is left at a crime scene can be compared with possible suspects to find out if the suspect was at the crime scene.
   

2. Medical: DNA profiles can be used to determine the parents of a child.
   

GENETIC SCREENING

Genetic Screening is used to find out if the parents of a child or the child itself (through removal of cells from the foetus) carry defective genes that could develop into health problems. Although the parent may not have the disorder he/she could be a carrier for the condition. With genetic screening, prospective parents can determine if there is a possibility that their future children could have the health problem.