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.

 

Is it Possible that DNA Genetic Material itself to fool the Definite Determination?           Part 5

Lawyers shall stand on their toes on floor to question the protocols used by laboratories, 

Eukaryotic cells heavily depended on noncoding codon triplets for their survival as well as to wither the environmental impacts through following biological functions. Some noncoding DNA is transcribed into functional non-coding RNA molecules (e.g. transfer RNA, ribosomal RNA, and regulatory RNAs). Other functions of noncoding DNA include the transcriptional and translational regulation of protein-coding sequences, scaffold attachment regions, origins of DNA replication, centromeres and telomeres.- defense system of genes as well as to preserve  the individuality of chromosome within the sets of chromosome in the cell nucleus. These telomere codons are common to all vertebrate animals, the sequence of nucleotides in telomeres is TTAGGG. This sequence of TTAGGG is repeated approximately 2,500 times in humans.  In forensic science these noncoding codon triplets are known as INTRONS, though in molecular biology and in genetics it covers the noncoding codon triplets as well as the all RNAs it can be transcribed into and the codons the RNA contains. In forensic and anthropological investigations, DNA introns are targeted for whatever procedure– RLFP or PCR or STR is applied for many reasons.

Using the noncoding introns for forensic science – was an accidental invention; researchers were working for variation for individuality amongst only 0.1% variant for human population for the entire globe.- since 99.9% of human DNA coding sequences are the same in every person, but enough of the DNA is different that it is possible to distinguish one individual from another, unless they are monozygotic ("identical") twins due to one fertilized egg blasting into two before going through embryonic transformation and both becoming two full embryos as normal.

Alec Jeffrey and members of his Forensic laboratory at the University of Leicester in the UK working with normal genes of codons RLFP analysis, suddenly found stretches of repetitive codons of DNA differ in length in the analytical output. Usual targets for analysis in the coding codons were of repetitive sequences known as Alleles – RLFP analysis of these alleles between two loci* are short repetitive and inconclusive due to mutations  (sudden favourable or unfavorable change of DNA sequences for living cell  during the process of replication of DNA) etc. compelling the workers to go for multiple loci analysis. *The specific location or position of a gene, on a chromosome known as locus (plural loci) – in DNA set of Triplet codon representing a gene.

But on that day analytical result of repetitive sequences of Alleles were of lengthy as well as surprisingly very conclusive as well, and later it was found they have performed analysis on a “wrong” target of noncoding introns – but for setting in motion a  miraculous corrective pathway for forensic biological science. These repetitive loci were used as targets for the first generation of genetic fingerprints using the most first DNA profiling analytical method, Restriction Fragment Length polymorphism (RFLP) of variable loci. A new era in criminal investigation had begun and use of Introns as analytical target became ethical as well.

The basic technique for Restriction Fragment Length polymorphism (RFLP)

1. fragmenting the  extracted of DNA from a biological  sample by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest.

2. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis. A method to segregate biochemicals via electrical charge.  

3. The separated fragments are then transferred to a nitrocellulose or nylon filter; this procedure is called a Southern blot. The DNA fragments within the blot are permanently fixed to the filter, and the DNA strands are denatured (disfigured)

4. Hybridization of the membrane to a labeled DNA probe; Radiolabeled probe molecules are then added that are complementary to sequences in the genome that contain repeat sequences. These repeat sequences tend to vary in length among different individuals and are called variable number tandem repeat sequences or VNTRs.

5. The probe molecules hybridize to DNA fragments containing the repeat sequences and excess probe molecules are washed away.

6. The blot is then exposed to an X-ray film. Fragments of DNA that have bound to the probe molecules appear as dark bands on the film.

This probe was not too specific, and therefore bound against multiple repetitive sites, and led into obtain the Traders’ Bar-Code pattern that many people know, and now identify, as a DNA fingerprint The more bands are shared between two individuals, the closer they are!

 

The banding pattern of a genetic fingerprint is nothing more than a bar code that is unique for just one person on earth—with the only exception of monozygotic twins who need to be distinguished by more advanced methods on the molecular level and thus various DNA analytical methods could be employed to obtain it.

Until around 1991, forensic biologists needed large amounts of high-molecular DNA for the multi-locus RFLP ‘fingerprints, practically this meant relatively that a large amount of biological samples had to be recovered. A further drawback in the early years was a lack of safe bio-statistics and laboratory standards.

Instead of further dealing with complex properties of multiple loci—size, bio-statistics and inheritance—laboratories now performs RFLPs of just one variable locus per probe as it was much easier to determine their fragment length. Since the ‘single locus’ probes could easily be washed off the nylon membrane, and then another probe for another ‘single’ locus could be hybridised against the RFLP, the information content of single-locus DNA typing remained high. The Southern blot technique is laborious, and requires large amounts of un-degraded sample DNA and very is time consuming in months as well. These early techniques have been succeeded by PCR-based assays.

Historically the dramatic change in forensic biolgy does not occur solely by the virtuous of analytical target of introns alone; but by the remarkable improvements in extraction of DNA from nearly any biological sample, no matter how small it is. DNA can now be extracted from practically any biological substance left at a scene of crime or an accident, including teeth, blood, sperm, saliva, bones, hair, urine and feaces, added to this list  is revolutionary ability, to recover DNA from inert objects that could have used by the suspects, that could lead to solve the crimes. Any laboratory dealing with DNA test should be competent enough in this aspect first and further most.   

Is it Possible that DNA Genetic Material itself to fool the Definite Determination? Part IV

Police detectives shall be smart enough to out-smart the Gangsters’ smart thinking for Gang- Raping and Murder

Collection of samples for DNA investigations

In very first of my series articles I discussed about sperm swimming to reach the ovulated ova at the top of uterus or further down – animal to animal this may vary – but the fertilized egg have to be implanted within the uterus(normally) for further development ensuring a next generation for that species. This is nature’s code survival of each species. 

The sperm of the all animals, particularly of mammals well adapted to swim and reach the ovum and as such sperm heads are perfect DNA preservation capsules protecting the nuclear DNA against the all environmental hazards of desiccation, such as by acid, alcohol, laboratory materials etc.  Thus semen material, found at the crime scene of rape, however contaminated it can be, should be an absolutely perfect material for pinpointing the identified or arrested suspects, - though all depends on the efficiency of the laboratory handles the job.  In this modern era if a laboratory sends negative reply for a judicial query on the basis of semen sample as of desiccated or contaminated, and that particular laboratory has no any value in judiciary and the state cannot sway from the responsibility of establishing or finding alternatives to fit into the modern day universal requirements regarding law enforcement against the heinous crimes.

It is truly laborious for any laboratory to type the semen samples that are even somewhat “fresh” from the victim who was gang raped and murdered. In such cases court shall give ample time to the laboratory (requested by the lab) if they have accepted the compounded mixed personal semen samples to pinpoint the each and every culprit.  

In this respect, the law enforcement personals have to act smart and efficiently with commitment to outsmart the assumption of the gang that the gang raping would confuse even the laboratory for final determination and supply and support the lab with additional crime scene collection, such as hair, empty bottles and cans of beverages, bottle lids that could have been mouthed by the suspects, smeared clothes, any objects that could have abraded the suspects, in a hurry to finish a desire, the crime for a thrill.  In one of the completely blind folded cases –in Germany a triple murder was solved through a wrist watch found at the scene many days after the crime had taken place. In many developed countries like Germany it is the police that do the entire lab work – in this particular case as soon they found the wristwatch they gave a simple wash that ensured collections of some cell debris – they were able to collect some epithelial cells of the skin from the grooves of wrist wind of the watch. In this particular case Bundeskriminalamt (BKA)—the German Federal Bureau of Investigation would have depended on a technique presently popular among the western nations including USA known as “GENETIC FINGER PRINTING” which is not available in Sri Lanka and also not (yet) ready for such a sophisticated system. But German Federal Bureau of Investigation is totally against this Genetic Finger Printing for lot bureaucratic involved.  

But what I emphasize here is sample resources for complete evidence that go with the collections of crime scene.   Sri Lanka is depended on RFLP analysis and PCR reaction test (which would be explained later), both of which are very effective as well if the lab work is so efficient to extract DNA needed—around 10 µg at least.   The same amount need for Genetic Finger Printing, as well which is nothing to do with the normal finger printing but rather with the Nucleus of the entire cells of the body, but like in fingerprinting  genetic information of the criminals are stored for future references.     

It should be noteworthy that any Colour reactions and staining that are used in criminal investigations, mostly to obtain fingerprint patterns (from real fingers) from a crime scene,  Surprisingly, neither superglue vapours nor any of these stains—amido black, leucomalachite green, Hungarian Red or luminol—interfere with DNA typing off the epithelial cells of entire skin. 

Is it Possible that DNA Genetic Material itself to fool the Definite Determination?           Part 3

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.

Control of gene expression

Last episode we were discussing about coding sequence of triplet codons and the non-coding sequences of Triplet Codons of the DNA molecules and termed the later as of ,  "useless" codons unless externally for forensic investigations. 

Based on the theory of evolution by Lamarck the first evolutionist by 1700

 “A life particle cannot maintain an abundant excess to be fittest for the best of survival”

Terming of non-coding sequences of Triplet Codons of the DNA as of "Junk", “useless” sequences is wrong biologically and though somewhat an older practice since as soon as the DNA was exposed as main component of chromosomes which was until then (1960) the only material for genomics of life studies.

As the name implies they do not encode for any proteins produced that ultimately decides the body shape, internal organs and metabolism. Later it was found that the genetically “useless” triplet codons transcribes into a noncoding transcribed transfer RNA, produces messenger RNA without a message and regulator ribosomal RNA etc. and brings about a halt to protein synthesis; unless otherwise the coding sequences for synthesis of proteins, would continue to synthesize proteins without any regulations harming the bodily functions and shape for the worst abnormalities within a life. It is postulated that non-coding sequences of Triplet Codons are actually functional as of all important regulating the protein synthesis within the body cell.

Synthesizing protein, Transcription and translation

There are many types of cells in a person’s body, such as heart cells, liver cells, and muscle cells. These cells look and act differently and produce very different chemical substances, though every cell is the descendant of a single fertilized egg cell and as such contains essentially the same DNA. Cells acquire their very different appearances and functions because different genes are expressed in different cells (and at different times in the same cell). The information about when a gene should be expressed is also coded in the DNA. Gene expression depends on the type of tissue, the age of the person, the presence of specific chemical signals, and environment and its mechanisms.

Proteins are composed of a long chain of amino acids linked together one after another. There are 20 different amino acids that can be used in protein synthesis—nine must come from the diet (9essential amino acids), and some are made by enzymes in the body. As a chain of amino acids is put together, it folds upon itself to create a complex three-dimensional structure. It is the shape of the folded structure that determines its function in the body. Because the folding is determined by the precise sequence of amino acids, each different sequence results in a different protein. Some proteins (such as hemoglobin) contain several different folded chains. Instructions for synthesizing proteins are coded within the DNA.

Transcription is the process in which information coded in DNA is transferred (transcribed) to ribonucleic acid (RNA). RNA is a long chain of bases just like a strand of DNA, except that the base uracil (U) replaces the base thymine (T). Thus, RNA contains triplet-coded information just like DNA.

When transcription is initiated, part of the DNA double helix splits open and unwinds. One of the unwound strands of DNA acts as a template against which a complementary strand of RNA forms. The complementary strand of RNA is called messenger RNA (mRNA). The mRNA separates from the DNA, leaves the nucleus, and travels into the cell cytoplasm (the part of the cell outside the nucleus. there, the mRNA attaches to a ribosome, which is a tiny structure in the cell where protein synthesis occurs.

With translation, the mRNA code (from the DNA) tells the ribosome the order and type of amino acids to link together. The amino acids are brought to the ribosome by a much smaller type of RNA called transfer RNA (trRNA). Each molecule of trRNA brings one amino acid to be incorporated into the growing chain of protein, which is folded into a complex three-dimensional structure under the influence of nearby molecules called chaperone molecules.

Thus it is obvious from the above that there should be a start codon which is ATG, followed by an openly readable stretch of 100 odd triplet codons that code for few amino acids of particular protein free of noncoding triplets or stop codons such as TAA, TAG, & TGA TCC and are the final in the stretch; start codon transcribed by mRNA and trRNA as UAG and the stop codons as UAA, UAG, UGA and UGG.  

No group of triplet codons assigned for one amino acid will not code for another; but start codon of RNA UAG codes for amino acid Methionine as well. Similarly stop codon UGG codes for amino acid Tryptophan as well. Both amino acids are essential amino acids and are the only coded by just one each triplet codon in DNA. 

How many molecules of DNA are there in one chromosome?

One chromosome is made of two chromatids; and each chromatid is a DNA molecule. Each DNA molecule is a double helix. So two DNA molecules per chromosome.

Total of 3.3 billion Nucleotide base Pairs per cell Nucleus in 46 Chromosomes that makes 3300 million divided 92 (46 X 2) and yields 35 million Nucleotide base Pairs per Chromosome; thus roughly should carry almost 10  million triplet codons representing 20,000 thousand genes and the rest are non-coding triplet base pairs. The entire set of coding triplet codon and non-coding triplet available living cell is known as Genome

We have learned the universal DNA activity of Eukaryotas, that is of animals and plants as a platform to understand the forensic determination of various issues, that will follow then and there, sexing of offspring also would follow as I wind up the series. 

Is it Possible that DNA Genetic Material itself to fool the Definite Determination? Part 2

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.  

DNA (deoxyribonucleic acid) is the cell’s genetic material, contained in chromosomes within the cell nucleus and mitochondria. Except for certain cells for example, sperm and egg cells and red blood cells), the cell nucleus contains 23 pairs of chromosomes. A chromosome contains many genes. A gene is a segment of DNA that provides the code to construct a protein. The DNA molecule is a long, coiled double helix that resembles a spiral staircase. In it, two strands, composed of sugar (deoxyribose) and phosphate molecules, are connected by pairs of four molecules called bases, which form the steps of the staircase. In the steps, adenine is paired with thymine and guanine is paired with cytosine. Each pair of bases is held together by a hydrogen bond. A gene consists of a sequence of bases. Sequences of three bases code for an amino acid (amino acids are the building blocks of proteins) or other information.

From the above drawn picture you can see the one and only Nucleus situated in the center of the tiny cell(10 - 30 µm are Most Eukaryotic animal cells of today and one µm micro meter is equal to 1 /1000 of a millimeter). Modern Molecular Biology  laboratories are (should be) competently resourced to handle those tiny cell and extract the exact nuclear DNA, targeting the centrally located Nucleus, without the contamination of extra-nucleus substance, scientifically known as cytoplasm  of a cell particularly the numerous number of Mitochondria; usually samples  collected are of mouth swabs, blood of known persons or  to be compared with that of under investigation sample. Both type samples will contain numerous cells intact. Anyhow  DNA are subjected for limited fragmenting and an efficient technician will realize whether he/she had typed  of different source DNA, because of the hyper activity of a Nuclear DNA instead of law profile activity of other DNA.  To understand this part we must go into details of DNA molecular structure and know what is meant by a term “SEQUENCE” in relation to DNA.

·         A gene is a segment of DNA containing the code used to synthesize a protein.

·         A chromosome contains hundreds to thousands of genes.

·         Every human cell contains 23 pairs of chromosomes, for a total of 46 chromosomes.

·         A trait is any gene-determined characteristic and is often determined by more than one gene.

·         Some traits are caused by abnormal genes that are inherited or that are the result of a new mutation.

Proteins are probably the most important class of material in the body. Proteins are not just building blocks for muscles, connective tissues, skin, and other structures. They also are needed to make enzymes. Enzymes are complex proteins that control and carry out nearly all chemical processes and reactions within the body. The body produces thousands of different enzymes. Thus, the entire structure and function of the body is governed by the types and amounts of proteins the body synthesizes. Protein synthesis is controlled by genes, which are contained on chromosomes.

The genotype is a person’s unique combination of genes or genetic makeup. Thus, the genotype is a complete set of instructions on how that person’s body synthesizes proteins and thus how that body is supposed to be built and function.

The phenotype is the actual structure and function of a person’s body. The phenotype typically differs somewhat from the genotype because not all the instructions in the genotype may be carried out (or expressed). Whether and how a gene is expressed is determined not only by the genotype but also by the environment (including illnesses and diet) and other factors, some of which are unknown.

The karyotype is the full set of chromosomes in a person’s cells.

Genes - DNA

Genes consist of deoxyribonucleic acid (DNA). DNA contains the code, or blueprint, used to synthesize a protein. Genes vary in size, depending on the sizes of the proteins for which they code. Each DNA molecule is a long double helix that resembles a spiral staircase containing millions of steps. We all know that 3.3 billion pairs of nucleotides base (pH more than 7) represent around 20,000 genes; thus it is obvious more than one such base pair code for a particular gene. But the 3.3 billion steps of the staircase consist of pairs of only four different types of molecules bases of nucleotides, namely Adenine, Thymine Cytosine and Guanine, and in each step, in any DNA always the base Adenine (A) pairs only with the base Thymine (T), or the base Guanine (G) can pair only with the base cytosine (C).

Coding

Information is coded within DNA by the sequence in which the bases (A, T, G, and C) are arranged. The code is written in triplets. That is, the bases are arranged in groups of three. Particular sequences of three bases in DNA code for specific instructions, such as the addition of one amino acid to a chain. For example, GCT (Guanine, Cytosine, and Thymine) codes for the addition of the amino acid alanine, and GTT (guanine, thymine, and thymine) codes for the addition of the amino acid Valine. Thus, the sequence of amino acids in a protein is determined by the order of triplet base pairs in the gene for that protein on the DNA molecule. The process of turning coded genetic information into a protein involves transcription and translation. The order of Triplet Base pairs in the Gene or DNA generally known as sequence. 

Above of all Nuclear DNA consists of numerous idling Nucleotide bases in pairs in its stairs and are known as non-coding sequences of Triplet Codons and technically called as Introns of DNA and these useless non-coding sequences  are the one used for forensic investigations and represents a staggering 93%  of a single DNA molecule. Theses non-coding sequences  are totally absent in Mitochondrial DNA