Basics of DNA & Evidentiary Issues Krishan Vij, Rajesh Biswas
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1Basic Concepts
An estimated 30,000 to 40,000 genes that encode more than ten times that number of proteins make up the human genome2

What is DNA?1

Each of the 100 trillion or more cells in a human being is a living structure that can survive indefinitely and, in most instances, can even reproduce itself. Before jumping onto the structure of deoxyribonucleic acid (DNA), it will be in the fitness of things to study the structure of the cell itself. A typical cell, has two major parts i.e. the nucleus and the cytoplasm. The nucleus is separated from the cytoplasm by a nuclear membrane, and the cytoplasm is separated from the surrounding fluids by a cell membrane. The different substances that make up the cell are collectively called protoplasm, which is composed mainly of five basic structures: water, electrolytes, proteins, lipids and carbohydrates. The cell is not merely a bag of fluid, enzymes and chemicals; it also contains highly organized physical structures many of which are called organelles. Five specially important organelles include: the endoplasmic reticulum, the Golgi apparatus, mitochondria, lysosomes and peroxisomes (Fig. 1.1).
 
 
Endoplasmic Reticulum
This is present in the cytoplasm in the form of a network of tubular and flat vesicular structures. The tubules and vesicles interconnect with one another. Their walls are constructed of lipid bilayer membranes that contain large amounts of proteins, similar to the cell membrane. Substances formed in some parts of the cell enter the space of the endoplasmic reticulum and are then conducted to other parts of the cell. Also, the vast surface area of the reticulum and multiple enzyme systems attached to its membranes provide the machinery for a major share of the metabolic functions of the cell.
 
Ribosomes and the Granular Endoplasmic Reticulum
Attached to the outer surfaces of many parts of the endoplasmic reticulum are large numbers of minute granular particles called ribosomes. Where these are present, the reticulum frequently is called the granular endoplasmic reticulum. The ribosomes are composed of a mixture of ribonucleic acid (RNA) and proteins, and they function in the synthesis of new protein molecules in the cell.4
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Fig. 1.1: Diagram of structure of a cell
 
Agranular Endoplasmic Reticulum
A part of the endoplasmic reticulum has not attached ribosomes. This part is called the agranular or smooth endoplasmic reticulum. The agranular reticulum functions in the synthesis of lipid substances and in many other enzymatic processes of the cell.
 
Golgi Apparatus
It is closely related to the endoplasmic reticulum as shown in the Figure 1.1. It has membranes similar to those of the agranular endoplasmic reticulum. It is usually composed of four or more stacked layers of thin, flat enclosed vesicles lying near one side of the nucleus. This apparatus is prominent in secretory cells. The Golgi apparatus functions in association with the endoplasmic reticulum. Small ‘transport vesicles’, also called endoplasmic reticulum vesicles or simply ER vesicles, continually pinch off from the endoplasmic reticulum and fuse with the Golgi apparatus. In this way, substances entrapped in the ER vesicles are transported from the endoplasmic reticulum to the Golgi apparatus. The transported substances are then processed in the Golgi apparatus to form lysosomes, secretory vesicles, or other cytoplasmic components.5
 
Lysosomes
These are vesicular organelles that form by breaking off from the Golgi apparatus and then dispersing throughout the cytoplasm. The lysosomes provide an intracellular digestive system that allows the cell to digest within itself (i) damaged cellular structures, (ii) food particles that have been ingested by the cell, and (iii) unwanted matter such as bacteria. It is surrounded by a typical lipid bilayer membrane and is filled with large numbers of small granules which are protein aggregates of different hydrolase (digestive) enzymes. Ordinarily, the membrane surrounding the lysosome prevents the enclosed hydrolytic enzymes from coming in contact with other substances in the cell. However, many conditions of the cell disturb the membranes of some of the lysosomes, allowing release of the enzymes. These enzymes then split the organic substances into small, highly diffusible substances such as amino acids and glucose.
 
Peroxisomes
These are similar to lysosomes, but differing in two important ways: First, they are believed to be formed by self-replication rather than by the Golgi apparatus. Second, they contain oxidases rather than hydrolases. Several of the oxidases are capable of combining oxygen with hydrogen ions from different intracellular chemicals to form hydrogen peroxide (H2O2), a highly oxidising substance which is used in association with catalase (another oxidase enzyme present in large quantities in peroxisomes), to oxidize many substances that might otherwise be harmful to the cell.
 
Mitochondria
These are called the “powerhouses” of the cell. Without them, the cells would be unable to extract significant amounts of energy from the nutrients which would be extremely detrimental to cellular functions. Basically, mitochondrion is mainly composed of two lipid bilayer-protein membranes: an outer membrane and an inner membrane. Many infoldings of the inner membrane form shelves onto which oxidative enzymes are attached. In addition, the inner cavity of the mitochondrion is filled with a matrix that contains large quantities of dissolved enzymes that are necessary for extracting energy from the nutrients. These enzymes operate in association with the oxidative enzymes on the shelves to cause oxidation of the nutrients, thereby forming carbon dioxide and water and at the same time releasing energy. The liberated energy is used to synthesize a high energy substance called adenosine triphosphate (ATP). The ATP is then transported out of the mitochondrion, and it diffuses throughout the cell to provide energy wherever it is needed for performing various functions of the cells.
Mitochondria are self-replicative, i.e. one mitochondrion can form a second one, a third one and so on, depending upon the need in the cell for increased amounts of ATP. Indeed, the mitochondria contain deoxyribonucleic acid (DNA) similar to that found in the nucleus. (DNA is the basic chemical of the nucleus and controls the replication of the cell. The DNA of the mitochondrion plays a similar role in the mitochondrion for controlling its own replication).6
 
Nucleus
Nucleus is the control center of the cell. If a cell is cut in half, the anucleate portion eventually dies without dividing. Majority of the DNA is located in the nucleus, organized in the form of chromosomes (22 pair of autosomes and a set of sex chromosomes, called X and Y). These chromosomes carry a complete blueprint for all the heritable species and individual characteristics of the individual. Except in germ cells, the chromosomes occur in pairs, one originally from each parent. The nuclear membrane is actually a bilayered envelope, one inside the other. The outer membrane is continuous with the endoplasmic reticulum of the cell cytoplasm, and the space between the two nuclear membranes is also continuous with the space inside the endoplasmic reticulum as shown in the Figure 1.2. The nuclear membrane is penetrated by nuclear pores. Large complexes of protein molecules are attached at the edges of the pores so that the central area of each pore is reduced considerably. The nuclei of most cells contain one or more structures called nucleoli. The nucleolus, unlike most other organelles, does not have a limiting membrane. Instead, it is simply an accumulation of large amounts of RNA and proteins of the types found in ribosomes.
Fertilization and Determination of Sex
Human cells (except the gamete cells, the egg and the sperm) contain 46 chromosomes. This chromosomal number 46 found in most somatic cells, is designated as diploid (2n). These 46 chromosomes are organized in pairs (23 pairs). Out of these, 22 pairs are morphologically similar and are called as autosomes.
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Fig. 1.2: Structure of a nucleus
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The 23rd pair is composed of the sex chromosomes, designated X and Y. In the female, it consists of two X chromosomes; in the male, the pair consists of an X and a Y. Each sex cell, as a result of meiosis, has only 23 chromosomes i.e. one member of each homologous pair. This number is designated as haploid(n). At the time of fertilization, the chromosomal number is again reestablished at 46, 23 from the egg and 23 from the sperm. An individual's complete chromosomal pattern as represented in a photograph is known as karyotype. Autosomal pairs, numbering from 1 to 22, are identified on the basis of their morphological characters. The human Y chromosome is smaller than the X and it has been suggested that sperms containing the Y chromosome are lighter and are able to ‘swim’ faster up the female genital tract, thus reaching the ovum more rapidly. A diagrammatic as well as descriptive account of various stages/processes leading to fertilization and determination of sex is furnished through Figures 1.3A to E.
Basics of Molecular Biology of DNA
DNA is sometimes referred to as the blueprint of life. The basic chemical compounds involved in the formation of DNA include:
  1. Phosphoric acid,
  2. A sugar called deoxyribose, and
  3. Four nitrogenous bases [two purines, adenine (A) and guanine (G), and two pyrimidines: thymine (T) and cytosine (C)]. The phosphoric acid and deoxyribose form the two helical strands that are the backbone of the DNA molecule and the bases lie between the two strands and connect them.
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    Fig. 1.3A: Male reproductive system
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    Fig. 1.3B: Spermatogenesis
    The specific sequence of the bases determines all the genetic attributes of a person. DNA in nature takes the form of a double helix (Fig. 1.4A). Two ribbon-like entities are entwined around each other and are held together by bonds, like rungs of the ladder. Each rung of two bases is called a base pair. Only specific pairings between the four bases will match-up and stick together. A always pairs with T, and G with C. This obligatory pairing, called complementary base pairing is exploited in all DNA typing systems. However, the term nucleotide denotes a base having one of four chemicals: Adenine (A), Thymine (T), Cytosine (C) or Guanine (G) as shown in Figure 1.4B. When the double helix is intact, the DNA is called double-stranded; when the two halves of the helix come apart, the DNA is called single-stranded. In nature, complementary base pairing is responsible for the ability to accurately replicate the DNA molecule with its genetic information, and pass it on to the next generation. Using each half of the original helix as a template, a second half is created, resulting in two molecules, identical to the original. This process can be recreated in vitro and is the basis of polymerase chain reaction (PCR). If the sequence at a particular location of the genome is of interest, single-stranded fragments can be artificially synthesized to target that location. These single-stranded fragments of known sequence are variously called as DNA probes or DNA primers depending on their intended use. [As per News Reports, recently researchers from six countries-Britain, China, France, Germany, Japan and the United States of America; succeeded in decoding all the chapters of the instruction-book for human life and reported that they had completed work on mapping human genome.
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    Fig. 1.3c: Composition of human semen
    (The term genome is used to describe complete collection of genetic information contained in the genetic regions/ segments and the extra DNA). As per their report, the complete sequence includes an estimated 30,000 to 40,000 genes that encode more than ten times that number of proteins].
The genomic DNA is composed of ‘coding’ and ‘non-coding’ regions (Fig. 1.4C). The coding regions are known as genes, which are programmed to provide information for a cell to make proteins.10
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Fig. 1.3D: Female reproductive system
Genes make up about 5% of the total human genomic DNA and the rest 95% consists of repetitive, primarily non-coding sequences of uncertain function, referred to as ‘junk DNA’. Interestingly, it is this junk DNA that shows variations amongst individuals and, therefore, utilized in DNA profiling for identification purposes. A particular molecular location is called a locus (plural being loci). Through scientific investigation, standard locations in the genome have been established where the sequence varies more than usual amongst people. The existence of multiple alleles of a marker at a single locus is called polymorphism. When such loci exhibit extreme numbers of variants, they are called hypervariable. Variations, or polymorphisms, can occur either in the sequence of bases at a particular locus or in the length of a DNA fragment between two defined end-points; called sequence polymorphisms and length polymorphisms respectively. Repetitive DNA can be divided into two classes: tandemly repetitive sequences and interspersed repeats. Tandemly repetitive DNA accounts for approximately 1/3 of repetitive DNA and is divided into two categories:
  1. minisatellite DNA, ranging from 15 to 35 base pairs repeated many times and
  2. short tandem repeats (STRs), much shorter than minisatellite DNA (3 to 7 base pairs repeat) which exhibit a high degree of variability in overall length; these regions are also called “microsatellites”.
A site/location of a chromosome that dictates a particular trait is called a gene. Genes are divided into two sections: the exons and the introns (Fig. 1.5). Exon represents the coding segment (segment that codes for a protein during translation) of the gene whereas introns as the non-coding segment which is stretched between each exon.11
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Fig. 1.3E: Maturation and fertilization of ovum
Genes may be short as of 100 base pairs or long as of a million base pairs. The term genetic marker implies a segment of DNA with specific physical location on a chromosome whose inheritance can be traced through a family because DNA segments that lie near each other tend to be inherited together.
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Fig. 1.4A: Double-helical structure of DNA, with adenine (A) bonding to thymine (T) and cytosine (C) to guanine (G) Phosphoric acid (p) and deoxyribose, a sugar(s), form the two helical strands that are the backbone of the DNA molecule and the nitrogenous bases lie between the two strands
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Fig. 1.4B: Nucleotide
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Fig. 1.4C: The backbone of each DNA strand is composed of alternating phosphoric acid and deoxyribose molecules. The nitrogenous bases (two purines; adenine and guanine, and two pyrimidines: thymine and cytosine) are attached to the deoxyribose molecules. This bondage is by means of loose hydrogen bonds and because of looseness of the hydrogen bonds, the two strands can pull apart with ease. The genetic code consists of successive “triplets” of the bases as shown in the figure
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Fig. 1.5: Showing two sister chromatids joined at centromere and a segment of genetic region
Hence, markers may be used as an indirect ways of tracking the inheritance pattern of genes from generation to generation.
Different forms of same gene or marker at a particular locus on a chromosome are called alleles. Different alleles produce variations in inherited characters such as hair 15or eye color and blood type, etc. ABO blood types are an example of different alleles of the same gene in humans. Genetic identity of an individual which does not show as outward characteristics is known as genotype i.e. it is inheritance pattern of a group of genetic markers for a particular individual. (if there are two alleles, ‘A’ and ‘a’ of a gene at a locus, there are three different genotypes-‘AA’, ‘Aa’ and ‘aa’). The term phenotype implies observable traits or characters of an organism. These traits may not necessarily be genetic and rather, in some way, expression of one's genetic make up plus the environment in which one grows and develops.
All genetic markers are found in the population with particular frequencies. For example, it is well known that ABO blood groups exhibit different frequencies in different populations. Each allele of the markers used in DNA analysis also exhibits a particular population frequency. It is important to determine the frequency with markers to attach a measure of strength to any particular genetic type.
Ordinarily, we have two copies of every gene in the genome, one copy on one member of a pair of chromosomes, and the other copy of the gene on the other member of the pair of a specific chromosome. When an individual carries two identical copies of a gene on his/her pair of a specific chromosome, he/she is homozygous for that gene. On the other hand, if two different variants of a gene are carried on a pair of chromosomes (one from the mother and one from the father), the individual is called heterozygous for that gene. The term genetic linkage implies association of genes that lie near each other on a chromosome. Linked genes tend to be inherited together as may be exemplified when a trait or a disease is inherited through a family and all in the family also share a genetic marker. Those situated far from each other on a chromosome or on separate chromosomes, are shuffled irregularly and are generally inherited independently of one another. This is called random assortment.