In which image will adenine (a) be the next nucleotide to be added to the primer?

Base pairing and hybridization of DNA strands

In its natural state in the cell, DNA is actually composed of two strands that are linked together through a process known as hybridization. Individual nucleotides pair up with their ‘complementary base’ through hydrogen bonds that form between the bases. The base-pairing rules are such that adenine can only hybridize to thymine and cytosine can only hybridize to guanine (Figure 2.2). There are two hydrogen bonds between the adenine–thymine base pair and three hydrogen bonds between the guanine–cytosine base pair. Thus, GC base pairs are stuck together a little stronger than AT base pairs. The two DNA strands form a twisted ladder shape or double helix due to this ‘base-pairing’ phenomenon (Figure 2.2).

The two strands of DNA are ‘anti-parallel’; that is, one strand is in the 5′ to 3′ orientation and the other strand lines up in the 3′ to 5′ direction relative to the first strand. By knowing the sequence of one DNA strand, its complementary sequence can easily be determined based on the base-pairing rules of A with T and G with C. These combinations are sometimes referred to as Watson–Crick base pairs for James Watson and Francis Crick who discovered this structural relationship in 1953.

Hybridization of the two strands is a fundamental property of DNA. However, the hydrogen bonds holding the two strands of DNA together through base pairing may be broken by elevated temperature or by chemical treatment, a process known as denaturation. A common method for denaturing double-stranded DNA is to heat it to near boiling temperatures. The DNA double helix can also be denatured by placing it in a salt solution of low ionic strength or by exposing it to chemical denaturants such as urea or formamide, which destabilize DNA by forming hydrogen bonds with the nucleotides and preventing their association with a complementary DNA strand.

Denaturation is a reversible process. If a double-stranded piece of DNA is heated up, it will separate into its two single strands. As the DNA sample cools, the single DNA strands will find their complementary sequence and rehybridize or anneal to each other. The process of the two complementary DNA strands coming back together is referred to as renaturation or reannealing.

13.1.1 DNA and its liquid crystalline phases

DNA is a semiflexible polymer made of two polynucleotide chains, held together by weak thermodynamic forces (Lander and Weinberg, 2000). The monomer units of DNA are the nucleotides. Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group (Travers and Muskhelishvili, 2015). There are four different types of nucleotides found in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). A and T are connected by two hydrogen bonds while G and C are connected by three hydrogen bonds (Fig. 13.1).

An interesting and comprehensively studied phenomenon is represented by the tendency of flexible polymers in concentrated aqueous solutions to form liquid crystalline phases (Onsager, 1949; Nakata et al., 2007). Indeed, the ability of both long and ultrashort, hydrated, double-stranded DNA molecules to form liquid crystal (LC) phases has been known for more than 50 years and played a key role in the initial deciphering of the molecular DNA. Linear DNA fragments in aqueous solution form multiple LC phases whose nature depends on the polymer concentration: when increasing concentration, the isotropic solution transforms into either blue phase or precholesteric stage and then into a cholesteric phase which turns itself into columnar hexagonal (Strzelecka et al., 1988).

Genetic Research Terminology

First, we provide some very brief background in the terminology of genetics for those who are not familiar with this field. The human genome consists of an ordered series of paired nucleotides in two matching strings, arrayed in a double helix as deoxyribonucleic acid, or DNA, in 23 chromosomes, or discrete strands of DNA. As shown in Figure 2, cells in the human body contain each of these 23 chromosomes. There are four nucleotides in the human genome: adenine (A), thymine (T), guanine (G), and cytosine (C). These are paired together determinatively, such that A is always paired with T and G with C. A polymorphism is a section of the genome in which variations in this base pair sequence are found between humans; a single nucleotide polymorphism (SNP) occurs when this consists of a single base pair difference. The different proteins are illustrated by the different colors in Figure 2. This variation in the human genome produces alleles, or DNA sequences which vary among humans. The observed combination of alleles at a location in one's genome is known as one's genotype at that location. Genetic researchers frequently seek to link genotypic variation to variation in phenotypes, which is the biological term for an organism's characteristics or traits. Phenotype is a synonym for ‘outcome variable’ in social scientific research.

Behavioral genetics is a field of behavioral science which attempts to summarize the separate contributions of genetic and environmental influences on a given phenotype. Frequently behavioral genetic researchers attempt to decompose this variance into three sources: (1) heritability, which describes the proportion of variance in an outcome that is thought to be explained by all genetic influences; (2) shared environments, which characterizes the environmental influences which make pairs of individuals more phenotypically similar; and (3) unshared environments, which characterizes the environmental influences which make pairs of individuals more phenotypically dissimilar. Behavioral genetics studies are frequently conducted using twin studies (which compares the phenotypic similarity of monozygotic twins, who are genetically identical, and dizygotic twins, who share 50% of their genes on average by descent) and adoption studies (which compares the phenotypic similarity of adopted sibling pairs to that of nonadopted members of their biological family).

When genetic information is available from subjects in a study, researchers can do two types of research. The first is called a candidate gene study in which individuals are classified into groups in which they are comparable to one another across segments of the human genome that are responsible for known physiological functions. These different groups are then compared with one another to see whether they are different with respect to some behavior like smoking, height, etc. There are many different statistical techniques for candidate gene studies, but using the correct method, researchers can conclude that the gene is actually responsible for differences in the trait and accounts for the similarity of family members who are much more likely to share the gene than two unrelated individuals. Genome-wide association studies (GWAS) incorporate data on a large number of SNPs (typically between 100 000 and 2 000 000 at this time) in order to identify loci associated with a phenotype of interest. Whereas the candidate gene method is a deductive exercise in which a specific hypothesis is tested, GWAS methods are entirely inductive and are much better characterized as a discovery science. In essence, GWAS models compare those with a specific illness (e.g., cancer) to those who do not have this illness (in which large efforts are made to provide the most meaningful comparisons) across each SNP in the human genome. Currently, genome-wide genotyping provide nearly 2.5 million SNPs. The strongest associations that emerge from this data mining exercise are then investigated to see whether they have any physiological pathway through which they might actually affect the trait of interest.

Genetic Markers and Repeated DNA Sequences

Because more than 99.7% of the human genome is the same from individual to individual, regions that differ need to be found in the remaining 0.3% in order to tell people apart at the genetic level. There are many repeated DNA sequences scattered throughout the human genome. Because these repeat sequences are typically located between genes, they can vary in size from person to person without impacting the genetic health of the individual.

Eukaryotic genomes are full of repeated DNA sequences. These repeated DNA sequences come in all types of sizes and are typically designated by the length of the core repeat unit and the number of contiguous repeat units or the overall length of the repeat region. Long repeat units may contain several hundred to several thousand bases in the core repeat.

These regions are often referred to as satellite DNA and may be found surrounding the chromosomal centromere. The term satellite arose because frequently one or more minor ‘satellite bands’ were seen in early experiments involving equilibrium density gradient centrifugation.

The core repeat unit for a medium-length repeat, sometimes referred to as a minisatellite or a VNTR (variable number of tandem repeats), is in the range of approximately 8 to 100 bases in length. As noted in Chapter 3, the previously used forensic DNA marker D1S80 is a minisatellite with a 16-bp repeat unit.

DNA regions with repeat units that are 2 to 7 bp in length are called microsatellites, simple sequence repeats (SSRs), or most usually short tandem repeats (STRs). STRs have become popular DNA repeat markers because they are easily amplified by the polymerase chain reaction (PCR) without the problems of differential amplification. This is because both alleles from a heterozygous individual are similar in size since the repeat size is small. The number of repeats in STR markers can be highly variable among individuals, which make these STRs effective for human identification purposes.

Literally thousands of polymorphic microsatellites have been characterized in human DNA and there may be more than a million microsatellite loci present depending on how they are counted. Regardless, microsatellites account for approximately 3% of the total human genome. STR markers are scattered throughout the genome and occur on average every 10,000 nucleotides.

Computer searches of the recently available human genome reference sequence have cataloged the number and nature of STR markers in the genome. A large number of STR markers have been characterized by academic and commercial laboratories for use in disease gene location studies. For example, the Marshfield Medical Research Foundation in Marshfield, Wisconsin (http://research.marshfieldclinic.org/genetics) has gathered genotype data on over 8000 STRs that are scattered across the 23 pairs of human chromosomes for the purpose of developing human genetic maps (see Chapter 17).

To perform analysis on STR markers, the invariant flanking regions surrounding the repeats must be determined. Once the flanking sequences are known, then PCR primers can be designed and the repeat region amplified for analysis (Figure 8.1). New STR markers are usually identified in one of two ways: (1) searching DNA sequence databases such as GenBank for regions with more than six or so contiguous repeat units or (2) performing molecular biology isolation methods. The availability of a reference human genome sequence now makes the first option a viable and productive one, and more than 20,000 tetranucleotide STR repeats have been located throughout the human genome. However, when the core STR loci that are widely used today were selected back in the mid-1990s, only a handful of STR loci were known and characterized.

STR allele nomenclature

To aid in interlaboratory reproducibility and comparisons of data, a common nomenclature has been developed in the forensic DNA community. DNA results cannot be effectively shared unless all parties are speaking the same language and referring to the same conditions. (It would do little good to describe the recipe for baking a cake in a language that is not understood by both the recipe giver and the chef. For example, if the recipe says to turn the oven on to 450°F and the chef uses 450 Kelvin [∼250°F], the results would be vastly different.)

If one laboratory calls a sample 15 repeats at a particular STR locus and the same sample is designated 16 repeats by another laboratory, a match would not be considered, and the samples would be assumed to come from separate sources. As will be discussed in Chapter 12, the advent of national DNA databases with many laboratories contributing information to those databases has made it crucial to have internationally accepted nomenclature for designating STR alleles.

A repeat sequence is named by the structure (base composition) of the core repeat unit and the number of repeat units. However, because DNA has two strands, either of which may be used to designate the repeat unit for a particular STR marker, more than one choice is available and confusion can arise without a standard format. Also, where an individual starts counting the number of repeats can also make a difference. With double-stranded DNA sequences being read in the 5′ to 3′ direction, the choice of the strand impacts the sequence designation. For example, the ‘top’ strand for an STR marker may be 5′-…(GATA)n…-3′ while the ‘bottom’ strand for the same sequence would be 5′-…(TATC)n…-3′. Depending on the sequence surrounding the repeat region, the core repeat could be shifted relative to the other strand (Figure 8.2).

Figure 8.2. Example of the DNA sequence in a STR repeat region. Note that using the top strand versus the bottom strand results in different repeat motifs and starting positions. In this example, the top strand has 6 TCTA repeat units while the bottom strand has 6 TGAA repeat units. Under ISFG recommendations, the top strand from GenBank should be used. Thus, this example would be described as having [TCAT] as the repeat motif. Repeat numbering, indicated above and below the sequence, proceeds in the 5′-to-3′ direction as illustrated by the arrows.

Allelic ladders

An allelic ladder is an artificial mixture of the common alleles present in the human population for a particular STR marker. They are generated with the same primers as tested samples and thus provide a reference DNA size for each allele included in the ladder. Allelic ladders have been shown to be important for accurate genotype determinations. These allelic ladders serve as a standard like a measuring stick for each STR locus. They are necessary to adjust for different sizing measurements obtained from different instruments and conditions used by various laboratories.

Allelic ladders are constructed by combining genomic DNA or locus-specific PCR products from multiple individuals in a population, which possess alleles that are representative of the variation for the particular STR marker. The samples are then coamplified to produce an artificial sample containing the common alleles for the STR marker (Figure 8.3). Allele quantities are balanced by adjusting the input amount of each component so that the alleles are fairly equally represented in the ladder. For example, to produce a ladder containing five alleles with 6, 7, 8, 9, and 10 repeats, individual samples with genotypes of (6,8), (7,10), and (9,9) could be combined. Alternatively, the combination of genotypes could be (6,9), (7,8), and (10,10) or (6,6), (7,7), (8,8), (9,9), and (10,10).

Figure 8.3. Principle of allelic ladder formation. STR alleles from a number of samples are analyzed and compared to one another. Samples representing the common alleles for the locus are combined and reamplified to generate an allelic ladder. Each allele in the allelic ladder is sequenced since it serves as the reference material for STR genotyping. Allelic ladders are included in commercially available STR kits.

Additional quantities of the same allelic ladder (second- and third-generation ladders) may be produced by simply diluting the original ladder 1/1000 to 1/1,000,000 parts with deionized water and then reamplifying it using the same PCR primers. It is imperative that allelic ladders be generated with the same PCR primers as those used to amplify unknown samples so that the allele ‘rungs’ on the ladder will accurately line up with that of the repeat number of the unknown sample when the unknown is compared to the ladder. Commercial manufacturers now provide allelic ladders in their STR typing kits so that individual laboratories do not have to produce their own allelic ladders.

Description of Genotypes and Phenotypes

As a result of the discoveries in molecular and cellular biology since Johanssen’s time genotypic and phenotypic descriptions have become more concrete, more detailed and greatly amplified in their scope. Originally genotypes had no independent physical descriptors but were distinguished from each other entirely on the basis of the external phenotypic manifestations and the patterns of similarity in phenotypes of related individuals. ‘Genes’ were simply abstract placeholders of unknown physical composition, inferred entities that could be characterized only by their phenotypic effects.

The modern identification of DNA as the physical material that behaves in heredity in the manner inferred for Mendel’s mysterious ‘factors’ has allowed a precise physical description of the genotype of an organism. It is the complete spatially-ordered sequence of the four alternative nucleotide bases, adenine, cytosine, guanine and thymine contained in the DNA of the organism’s cells. In its most complete form this genotypic description includes not only the DNA in the nucleus of the cell but in certain cellular organelles such as mitochondria or chloroplasts that carry the DNA that is related to their physiological function and that is passed across generational lines. Phenotypic description has also been greatly amplified beyond the external characteristics of individuals. It now includes the gross structure of the various cells of the organism, the physico-chemical description of the various molecules within cells, including their shape and intra-cellular localization, and the network of biochemical reactions in which these molecules participate.

The concepts of phenotype and genotype require a distinction between types and tokens. Phenotype and genotype are, as the words indicate, types, sets of which individual organisms are members, sets delineated by the complete descriptors of those organisms. Any individual member of these type sets is a token of the type. In addition it is necessary to distinguish the descriptors of organisms from the physical object or behavior that is being described. The concepts phenotype and genotype refer to the descriptors. The physical objects that are being described are the phenome and genome of an organism, the actual physical material or behavior of some particular individual and its actual DNA molecules. If we take the definitions literally, then no two organisms belong to the same phenotype because there is always some physical difference in morphology, physiology and behavior between any two organisms, even between identical twins, cloned individuals, or cells that result from asexual reproduction. Moreover, except for the results of twinning, cloning or vegetative reproduction, no two individuals belong to the same genotype, as the process of mutation guarantees some variation in their DNA between any two individuals.

The rate of mutation per nucleotide per cell division is of the order of one in a billion, so for mammals, with over a billion nucleotides of DNA making up their genes, even identical twins are likely to have some small difference in total genotype. There is, in addition, an ambiguity in the assignment of an individual to a genotype because mutations occur during the millions of cell divisions that occur in development, so that all cells in the body do not have identical DNA.

In practice phenotypic and genotypic descriptions are not total but partial, referring to some aspect of the genome and phenome that are of particular interest, while ignoring the rest. The partial phenotypic and genotypic descriptions are partial in two senses. First, only some aspect of the morphology or function of the organism is characterized; say the rate of enzymatic splitting of alcohol, a phenotype, and the DNA sequences that code for the enzyme proteins involved in the reaction, a genotype. Second, a decision must be made about the collection of actual partial phenotypes and genotypes that will be regarded as indistinguishable. At the phenotypic level, given the actual variation in enzymatic rates from individual to individual and from time to time, what range of rates will be regarded as belonging to the same phenotypic class? For any phenotypic variable that is continuous, some boundaries of distinguishable phenotypic classes must be laid down in order to make a mapping between phenotype and genotype because genotypes, by their nature, are discrete classes.

The problem of aggregation exists at the genotypic level as well. The relation of DNA differences to differences in the amino acid sequence of a protein is not one-to-one. Because the DNA triplet code is redundant, substitutions of nucleotides in the third position of the triplet often do not change the amino acid that is specified, so for some purposes many different alternative DNA sequences of a gene count as the same genotype. On the other hand, nucleotide substitutions that do not affect the amino acid sequence of a protein may affect the rate of synthesis of that protein and so may, for some questions, be counted as belonging to different genotypes.

The decision about which aspects of total phenotype and genotype are relevant in creating genotypic and phenotypic classes is a deep question of biology that is largely ignored because everything cannot be considered at once. Sometimes biologists act as if the rest of the organism really were constant, sometimes they recognize the variation but claim that it is causally irrelevant to the phenomenon of interest and sometimes they admit its causal relevance but claim that the variation can be treated as background experimental noise that can be averaged out by repeated observations on a large class of individuals. These decisions are usually made either for the sake of observational convenience without a coherent rationale or from a commitment to a reductionist view of the organism that asserts that an understanding of the whole system can be obtained by breaking it down into ‘obvious’ small components to be studied in isolation.

3.4.1 Flat solid surfaces

Gold electrodes. One interesting example is the self-assembled monolayer formation of a series of thiophene dendron thiols with different generations and alkyl chain lengths onto gold surfaces [60]. The purpose of the cited study was to understand the chemisorption behavior of a series of hyperbranched thiols formed onto gold electrodes in terms of parameters such as size, alkyl chain length, and bulk concentration. The chemical structures of these dendron thiols are shown in Fig. 3.6A. A rearrangement adsorption/desorption kinetics to describe their behavior at the interface was elucidated by using surface plasmon resonance, electrochemistry, quartz crystal microbalance and water contact angle measurements [60]. An empirical three-step model explained the observed adsorption kinetics, which involved a two-step rearrangement process including a relatively faster short range and a much slower long range course, followed by an initial fast adsorption/desorption. For the lower generation dendron thiols, much faster adsorption rate constants were found compared to higher generation dendron thiols, due to the additional adherence of the thiophene sulfurs to gold surfaces. Dendrons with the longest alkyl chain showed the most tightly packed monolayer arrangement [60]. Another focally substituted organothiol dendron shown in Fig. 3.6B) also adlayers on gold by chemisorption of the thiol moiety onto this surface [61]. The covalent approach onto gold surfaces is also achieved with sulfides at the focal point as shown by Friggeri et al., who investigated the insertion process of individual dendron-sulfide molecules (Fig. 3.6C) into self-assembled monolayers of 11-mercapto-1-undecanol by atomic force microscopy, wettability, and electrochemical measurements [62]. A mechanism consisting of rapid dissociation of surface thiols followed by slow dendron adsorption was proposed considering the insertion of the individual dendron molecules as the rate-determining step of the process. The immersion of alkanethiol self-assembled layers (SAM) in solutions of increasing concentrations of dendron-sulfide leads to an increase in the number of dendritic molecules inserted into the thiol layer; on the contrary, the initial SAM quality (reached at different incubation times of alkanethiols) is not a determining factor for the dendron insertion process.

Dendrons with a carboxylic acid at the focal point [3,5-bis (3,5-dinitrobenzoylamino) benzoic acid] and nitro groups at the periphery (G1-NO2) were immobilized onto gold surfaces in a noncovalent approach and studied by electrochemistry and scanning tunneling microscopy (STM) [63]. G1-NO2 adsorbs onto gold electrode surfaces spontaneously by dipping the metal surface in dendron solution and also via grafting of cystamine covalently attached to a gold electrode. The reduction of these layers exhibits a well-behaved redox response for the adsorbed nitroso/hydroxylamine couple, useful for the electrocatalysis of reduced nicotine adenine dinucleotide (NADH), an important feature in amperometric sensors design [63].

Dong et al. reported the formation of special surface structures on gold arising from the precisely tailored structure of surface-bound thiol-dendrons with different main structures and peripheral substituents [64]. Among the factors controlling aggregation behavior, chemisorption is fairly similar in these compounds. Thus, intermolecular interaction greatly influences the configuration of dendrons on gold and different results were obtained from SAMs composed of symmetrical and asymmetric structures (Fig. 3.7). The different aggregation behavior observed was probably influenced by peripheral substituents and the coadsorption process, as reflected in the results of both STM and electrochemical measurements [64].

A library of compact and monodisperse dendritic scaffolds based on the nontoxic 2,2-bis(methylol)propionic acid (bis-MPA) was explored for binding SAM onto gold surfaces to exploit the design of hydrophilic dendritic structures bearing sulfur-containing core functionalities [65]. The size of the dendritic framework (G1–G3), the nature of the sulfur (whether thiol or disulfide), the functional end group (mannose or hydroxyl), and the distance between the dendritic wedge and the sulfur were key structural elements affecting the packaging densities assembled on the substrates. Surface interactions between multivalently presented motifs and cells in a controlled surface setting were evaluated by the cell scavenging ability of these sensor surfaces for Escherichia coli Ms7fim+ bacteria that revealed 2.5-fold enhanced recognition for G3-mannosylated surfaces compared to G3-hydroxylated SADM surfaces [65].

Dendritic polyglycerol (PG) derivatives with different numbers of amino groups have been attached onto gold substrates via thioctic acid linker and the selective interaction of complementary fluorescently labeled DNA proved the availability of such end groups for biomolecule attachment. These results demonstrate a new way to tailor hyperbranched surfaces by introducing amino moieties which can act as suitable anchoring sites for specific biomolecule interactions, while maintaining the resistant properties against non-specific protein adhesion. The protein-resistant properties of these PG-coated surfaces depend on the amino content, providing a combination of both important characteristics in bioelectronics or in the development of biosensing platforms with improved sensitivity [1,2].

Carbon surfaces. The functionalization of carbon surfaces with dendrons provides controllable properties for the electrode surface due to multifunctional groups of these molecules. For example, the cooperative effect of phenyl rings and the multifunctionality of G1-NO2 and G2-NO2 dendrons (Fig. 3.8) allow a direct, rapid and spontaneous physisorption of these dendrons onto carbon surfaces [66]. The AFM images show a network film with embedded aggregates that completely cover the carbon surfaces after only a few minutes, with average heights suggesting a tilted preferred adsorption in the early stages of the film formation, and highlighting a noticeable increment of this effect with increasing dendron generation. These molecules form a layer covering the whole surface, but do not block the electron transfer reaction of redox probes like Fe(CN)63−/4− or Ru(NH3)63+/2+. This effect, together with the remarkable simplicity of obtaining nitroaryl-ended films, makes these modified electrodes promising for electrocatalysis and biosensing platforms [67].

Boltorn H30 molecules are the third generation of commercial polyhydroxylated hyperbranched polymers, are approximately spherical in shape, have an average diameter larger than 3 nm and inner cavities that can be used for small molecule or ion inclusion. The spontaneous adsorption of Boltorn H30 onto carbon substrates was carried out with the idea of generating a nanostructured layer capable of retaining copper cations (II) inside. The modified electrode was subsequently incubated for a certain length of time (between 1 and 3 h) in CuCl2 solutions, washed in water and taken to an electrochemical cell to reduce the cations and generate the metallic nanoparticles [68]. These platforms are valuable for the electrocatalysis of hydrogen peroxide.

Silicon surfaces (Si wafers in air are covered with a thin SiO2 layer bearing OH groups as final functions). Monodisperse dendrons with urea/malonamide are used as linkages for organic thin-film transistor gate insulators [69]. These dendrons are hydrogen bond-rich and able to form stable self-assembled structures. Due to their strong intermolecular interactions, they have been successfully employed in nonlinear optics. In addition, dendrons with hydrogen bond-rich urea/malonamide linkages and peripheral long alkyl chains would certainly interact with tetracarboxylic diimide derivatives possessing perfluorinated alkyl chains on imide rings [69].

Indium tin oxide (ITO) electrodes (transparent conducting films). Recently, new hyperbranched p-conjugated macromolecules were obtained by the versatile electropolymerization method and exhibit interesting electronic properties that yield thin and stable layers with good electrical conductivity. Mangione et al. reported the synthesis and properties of peripherally carbazole (CBZ) functionalized starburst monomers, featuring the presence or absence of electroactive central core triphenylamine (TPA) moieties connected by conjugated or saturated branches. For this purpose, the dendron monomers were obtained by a convergent strategy and the CBZ residues allowed the formation of hyperbranched polymeric layers over conductive substrates by electrochemical polymerization. The radical cation coupling of oxidized CBZ leads to the growth of the dendritic structures. Thus, two different fully p-conjugated dendritic polymers are formed, with and without an electroactive central core connected to peripherals moieties (Fig. 3.9) [70].

3.2 Human DNA that differs among individuals

The majority of human DNA is encapsulated in organelles called cell nuclei; this is called genomic DNA. Genomic DNA consists of four bases (adenine, thymine, guanine, and cytosine), and the arrangement of these bases specifies all the information necessary for human survival. An attempt to decode the whole genomic DNA sequence started in the United States in 1990 as the “Human Genome Project,” [7,8] and the entire sequence was determined in 2003 [9]. Human genomic DNA is composed of 3 billion base pairs, and about 0.1% of the sequence differs among individuals. As a result of detailed investigations on the sequences that differ among individuals in the HapMap project that began in 2003 [10] and in the 1000 genomes project that began in 2008 [11], their positions, sequence type, and their frequencies have become clear. When performing individual identification using DNA, analysts use sequences in which differences are often identified rather than sequences in which individual differences are observed very rarely. These genomic sequence differences with a frequency in the population of 1% or more are called “polymorphisms” and are distinguished from “mutations” with a frequency of 1% or less. The sequence used for individual identification is “polymorphism”.

There are various types of DNA polymorphisms, including single nucleotide polymorphism (SNPs), repetitive sequence polymorphism, insertion deletion polymorphism (Indel), and copy number polymorphism (CNV) [12].

The genetics and neurobiology of ADHD

The precise etiology of ADHD is not known, but numerous family, twin, and adoption studies reveal a strong genetic component. Heritability estimates for ADHD average about h2 = .80 and are consistently found regardless of whether it is considered a categorical or continuous trait (Bobb, Castellanos, Addington, & Rapoport, 2005). Behavioral genetic designs that produce heritability estimates show only that genes are involved in the etiology of ADHD, but not which genes; molecular genetics is required to do that. Molecular genetic studies show ADHD to be highly polygenic, with at least 50 genes with small to moderate effects being involved (Comings et al., 2005). The highly polygenic nature of ADHD probably explains why it is so clinically heterogeneous and why it is linked to many other problems such as CD, ODD, substance abuse, pathological gambling, and antisocial behavior in general.

The hunt for candidate genes implicated in any disorder involves looking at genetic polymorphisms. Polymorphism refers to the differences in allelic combinations (an allele is an alternate form of a gene; one inherited maternally and one paternally) that make us different from one another even though we share the same genes. There are three different types of polymorphisms: single nucleotide polymorphisms (SNPs), minisatellites and microsatellites (Altukhov & Salmenkova, 2002). As the term SNP implies, a difference in just one nucleotide is all that differentiates one allele from another. A nucleotide is one of the four nitrogenous bases (adenine, cytosine, guanine, thymine or uracil) plus a sugar and phosphate backbone that make up DNA and RNA. When we consider that the human genome contains approximately 3 billion base pairs we can appreciate the potential number of polymorphisms it contains. It is estimated that about 85% of genetic causes of most behavioral disorders are attributable to SNPs, the most frequently occurring polymorphism (Plomin et al., 2001). The other types are often lumped together as VNTRs (variable number of tandem repeats) that consist of sections of DNA repeated a different number of times in different alleles. We limit ourselves to the three polymorphisms that meta-analyses (Bobb et al., 2005; Gizer, Ficks, & Waldman, 2009) have shown to be most replicated in ADHD genetic studies and which are also implicated in criminal and other forms of antisocial behavior: the dopamine receptor D4 (DRD4), the dopamine transporter (DAT1), and the serotonin transporter (5-HTTLPR). We emphasize that genes associated with complex disorders such as ADHD are likely to have low penetrance and thus many carriers will not develop the disorder.

Whatever the genes associated with ADHD, their function is to manufacture neurotransmitters, receptors, transporters, or enzymes. These substances are utilized in all parts of the brain, but the most salient part for understanding ADHD is the prefrontal cortex (PFC), “the most uniquely human of all brain structures” (Goldberg, 2001, p. 2). This vital area has extensive connections with other cortical regions as well as with deep structures in the limbic system. Because of its many connections with other brain areas, it is generally considered to play the major integrative, as well as the major supervisory role in the brain, and is vital to the forming of moral judgments, mediating affect, and for social cognition (Romaine & Reynolds, 2005). It is the last part of the brain to mature—to become fully myelinated—which typically does not occur until mid-adolescence-early adulthood (Sowell et al, 2004). Myelin is the fatty substance that coats the neuronal axons and acts as insulation. Myelinated axons allow for speedier transmission of the electric message along the length of the axon. Not coincidentally, it is at this time that many ADHD symptoms especially impulsivity tend to subside. Although it is important to note that about 90% of ADHD individuals continue to exhibit some symptoms into adulthood (Willoughby, 2003).

ADHD can also be understood as a disorder of neurological regulation and underarousal, making the PFC a key focus of neuroscientists researching ADHD. The PFC provides us with knowledge about how others see and think about us, thus moving us to adjust our behavior to consider their needs, concerns, and expectations. These PFC functions are collectively referred to as executive functions (Tripp & Wickens, 2009), and are clearly involved in prosocial behavior if functioning normally. Beaver, Wright, and DeLisi (2007) have shown that deficits in neuropsychological functioning in various areas of the PFC are related to self-control levels and suggest that this revered criminological concept is an executive function. Meta-analyses of brain imaging studies show that reductions in the gray matter volume in the both left and right cortices of the PFC are the most robust and consistent brain region deficits associated with ADHD (Valera et al., 2007). The PFC requires optimal levels of the neurotransmitters dopamine and norepinephrine to function well. Too little dopamine and norepinephrine and we become drowsy, bored, or fatigued; too much and we are stressed, either of which impairs PFC functioning. In short: “Imaging studies have demonstrated that patients with ADHD have alterations in PFC circuits and demonstrate weaker PFC activation while trying to regulate attention and behavior” (Arnsten, 2009, p. 22).