Pathophysiology CH. 4 Genetic Control of Cell Function and Inheritance

Genetic Control of Cell Function and Inheritance

• Genetic information is stored in deoxyribonucleic acid (DNA), a very stable macromolecule.
• DNA directs cell function, appearance, response to the environment, and inheritance.
• Genotype influences disease susceptibility and drug reactions.
• The Human Genome Project (completed in 2003) sequenced nearly the entire human genome.
• Many common diseases like cancer, diabetes, and cardiovascular disease have genetic components.
• Recombinant DNA technology enables the production of human insulin, growth hormone, and clotting factors.
• The first end-to-end human genome was published in 2022.

Genetic Control of Cell Function

• DNA's stability ensures the survival of genetic information during cell division, renewal, and tissue growth.
• It also survives reduction division in gamete formation, fertilization, and mitotic divisions in zygote formation.
• Ribonucleic acid (RNA) is involved in protein synthesis.
• Information is transcribed from DNA to RNA, processed in the nucleus, and translated into proteins in the cytoplasm.
• Proteins form the majority of cellular structures and perform most life functions.

The Role of DNA in Controlling Cell Function

• Genetic information for cell structure and function is encoded within the DNA molecule.
• Each cell uses only a portion of the total genetic information, specific to its function.

DNA Structure and Function

• DNA is a long, double-stranded, helical molecule composed of nucleotides.
• Nucleotides consist of phosphoric acid, deoxyribose (a five-carbon sugar), and a nitrogenous base.
• Nitrogenous bases are divided into:
  • Pyrimidines: thymine (T) and cytosine (C) (one nitrogen ring)
  • Purines: adenine (A) and guanine (G) (two nitrogen rings).
• The DNA backbone consists of alternating sugar and phosphate groups.
• Base pairing rules: Adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C).
• The double-stranded structure of DNA allows for precise replication and repair.
• Before cell division, the DNA strands separate, and a complementary strand is duplicated alongside each original strand.
• During cell division, each daughter cell receives a pair of double-stranded molecules, each containing one old and one new strand.
• Meselson and Stahl (1958) characterized DNA replication as semiconservative.
• Semiconservative replication: original DNA strands unwind, and a complementary strand forms along each.
• Conservative replication: Original parental strands reassociate after replication.

Packaging of DNA

• The genome is distributed in chromosomes.
• Human somatic cells have 23 pairs of chromosomes (one from each parent).
• 22 pairs are autosomes; the 23rd pair are sex chromosomes (XX for female, XY for male).
• Genes are arranged linearly along each chromosome.
• Each chromosome contains one continuous, linear DNA helix.
• DNA molecules are combined with proteins and small amounts of RNA into chromatin.
• Histones control the folding of DNA strands.
• Tight compaction allows the large amount of DNA to fit into the nucleus and enables faithful replication during cell division.
• Coiling prevents certain genes from being accessed when not needed.
• Chromatin remodeling: the process of altering chromatin structure to allow access to specific genes.
• Acetylation of histone proteins triggers gene activation.
• Methylation of other histone proteins correlates with gene inactivation.

DNA Repair

• Mutations are accidental errors in DNA replication, resulting from base pair substitutions, loss/addition of base pairs, or rearrangements.
• Mutations can be spontaneous or caused by environmental agents, chemicals, and radiation.
• Mutations in germ cells can be inherited.
• DNA repair mechanisms correct most defects using specific enzymes called endonucleases.
• Endonucleases recognize distortions, cleave the abnormal chain, and remove the distorted region.
• DNA polymerase fills the gap using the intact complementary strand as a template.
• DNA ligase joins the newly synthesized segment to the remainder of the DNA strand.
• Specific DNA repair genes regulate these mechanisms; changes in these genes can lead to the accumulation of mutations and cancer.

Genetic Variability

• The human genome sequence is 99.9% identical across individuals.
• Small variations (0.01%) account for individual differences in physical traits, behaviors, and disease susceptibility.
• These normal variations are called polymorphisms.

From Genes to Proteins

• RNA is responsible for assembling amino acids into functional proteins in the ribosome through translation.

RNA Structure and Function

• RNA is a large, single-stranded molecule made up of nucleotides.
• Three key structural differences from DNA:
  1. Single-stranded vs. double-stranded.
  2. Ribose sugar instead of deoxyribose.
  3. Uracil (U) replaces thymine (T).
• Three types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
• All three types are synthesized in the nucleus by RNA polymerase enzymes and then moved into the cytoplasm for protein synthesis.

Messenger RNA (mRNA)

• mRNA carries instructions for protein synthesis from DNA to the cytoplasm.
• Four bases (guanine, adenine, cytosine, uracil) form the alphabet of the genetic code.
• A sequence of three bases in RNA forms a codon (triplet code).
• Example: UGG codes for tryptophan.
• The genetic code is universal across living cells.
• Four bases can be arranged in 64 different combinations.
• 61 triplets correspond to specific amino acids, and 3 are stop codons.
• The genetic code is redundant or degenerate because several triplets can code for the same amino acid.
• Codons specifying the same amino acid are synonyms, usually with the same first two bases but differing in the third base (wobble).
• mRNA is formed from DNA by transcription.
• Weak hydrogen bonds of DNA nucleotides are temporarily broken, allowing free RNA nucleotides to pair with their DNA counterparts.
• Guanine pairs with cytosine; uracil (U) pairs with adenine.

Ribosomal RNA (rRNA)

• The ribosome (made of rRNA and proteins) is the site of protein synthesis in the cytoplasm.
• rRNA is synthesized in the nucleolus.
• rRNA combines with ribosomal proteins in the nucleus to form the ribosome, which is then transported into the cytoplasm.
• Most ribosomes attach to the endoplasmic reticulum to facilitate protein synthesis.

Transfer RNA (tRNA)

• tRNA is a clover-shaped molecule that delivers the activated form of an amino acid to the ribosome.
• At least 20 different types of tRNA exist, each specific to one amino acid.
• Each tRNA molecule has two recognition sites: one complementary to the mRNA codon, and another complementary to the amino acid itself.
• tRNA delivers its specific amino acid to the ribosome, recognizes the codon on the mRNA, and adds the amino acid to the growing protein.

Transcription

• Transcription occurs in the cell nucleus.
• RNA is synthesized from a DNA template.
• Genes are transcribed by RNA polymerases, generating a single-stranded RNA identical in sequence (with the exception of U in place of T) to one of the strands of DNA.
• Transcription begins with the assembly of a transcription complex, composed of RNA polymerase and other associated factors.
• The complex binds to double-stranded DNA at the promoter region.
• The promoter region contains a TATA box (thymine-adenine-thymine-adenine nucleotide sequence) that RNA polymerase recognizes and binds to.
• Transcription factors, a transcription initiation site, and other proteins are required for this binding.
• Transcription continues, copying one strand of DNA into RNA until it reaches the stop codon.
• On reaching the stop signal, the RNA polymerase enzyme detaches and releases the RNA strand.
• The RNA strand is then processed into a mature mRNA molecule, involving the addition of nucleic acids at the ends and splicing of internal sequences.
• Splicing involves the removal of stretches of RNA called introns.
• The retained protein-coding regions are called exons.
• Splicing permits a cell to produce a variety of mRNA molecules from a single gene, reducing the amount of DNA needed in the genome.

Translation

• After processing, mRNA is transported to the cytoplasm, where translation occurs.
• Translation is the synthesis of a protein using the mRNA template.
• Proteins are made from amino acids joined end to end to form polypeptide chains.
• Translation requires the coordinated actions of mRNA, rRNA, and tRNA.
• mRNA provides the information for placing amino acids in the correct order.
• During protein synthesis, mRNA binds to rRNA in the ribosome, which “reads” the directions.
• tRNA delivers the appropriate amino acids for attachment to the growing polypeptide chain.
• Each of the 20 different tRNA molecules transports its specific amino acid to the ribosome.
• As each amino acid binds to the next by a peptide bond, its tRNA is released.
• The new polypeptide chain must then fold into its unique three-dimensional conformation.
• Molecular chaperones are special proteins that make the folding of many proteins more efficient.
• They also assist in transporting the protein to its functional site in the cell and prevent misfolding.
• Disruption of chaperoning mechanisms leads to denatured and insoluble proteins, forming inclusion bodies (a pathologic process in diseases like Parkinson, Alzheimer, and Huntington).
• Other modifications may include polypeptide chain combination, binding of small cofactors, and enzyme modification.
• Cleavage of the protein may also occur, removing specific amino acid sequences or splitting the molecule into smaller chains.

Regulation of Gene Expression

• Only about 2% of the genome encodes instructions for protein synthesis; the remainder consists of noncoding regions that are structural or serve to determine where, when, and in what quantity proteins are made.
• Gene expression refers to the degree to which a gene or group of genes are actively transcribed.
• Induction is the process by which gene expression is increased.
• Gene repression is the process by which gene expression is reduced or prevented.
• Activator and repressor sites commonly monitor synthesized product levels and regulate gene transcription through a negative feedback mechanism.
• Control of gene expression often occurs at the transcription level.
• Transcription factors are proteins that bind to specific DNA regions and increase or decrease transcriptional activity.
• Differences in transcription factors between cells and tissues allow all cells to use the same DNA yet still have completely different structures and functions.
• General transcription factors are required for transcription of all genes, while specific transcription factors have more specialized roles.
• Example: The PAX family of transcription factors is involved in the development of embryonic tissues like the eye and nervous system.

Understanding the Nucleic Acids: DNA and RNA

• DNA contains the information to direct the synthesis of the many thousands of proteins that are contained in the different cells of the body.
• RNA participates in the actual assembly of the proteins through the processes of transcription (interpreting the DNA instructions for protein synthesis) and translation (using those instructions to assemble the polypeptides that make up the various proteins).
• The genetic code is made up of four bases (adenine [A], thymine [T], guanine [G], and cytosine [C]). In RNA, the thymine base is replaced with uracil [U].
• Transcription occurs when the DNA is copied into a complementary strand of mRNA.
• Transcription is initiated by an enzyme called RNA polymerase, which binds to a promoter site on DNA.
• Many other proteins, including transcription factors, function to increase or decrease transcriptional activity of the genes to make more or less of the gene product.
• After mRNA has been transcribed, the strand detaches from DNA and is processed by adding nucleotide sequences to the beginning and end of the molecule, and introns are spliced out.
• Changes to the splicing allow the production of a variety of mRNA molecules from a single gene.
• Once mRNA has been processed, it diffuses through the nuclear pores into the cytoplasm, where it is translated into protein.
• Translation begins when the mRNA carrying the instructions for a particular protein comes in contact with a ribosome and binds to a small subunit of the rRNA.
• It then travels through the ribosome while the tRNA delivers and transfers the correct amino acid to its proper position on the growing peptide chain.
• The tRNA binds to a three-base section of the mRNA called a codon.
• There are 20 types of tRNA, one for each of the 20 different types of amino acid.
• In order to be functional, the newly synthesized protein must be folded into its functional form, modified further, and then routed to its final position in the cell.

Chromosomes

• Most of the DNA in a cell is organized, stored, and retrieved in structures called chromosomes.
• Although the chromosomes are visible only in dividing cells, they retain their integrity between cell divisions.
• The chromosomes are arranged in pairs where one member of the pair is inherited from the biological father and the other member is inherited from the biological mother.
• Each species has a characteristic number of chromosomes. In the human, 46 chromosomes are present, and these are arranged into 23 pairs.
• Of the 23 pairs of human chromosomes, 22 are called autosomes, and each has been given a numeric designation for classification purposes.
• The pairs of autosomal chromosomes are called homologous chromosomes.
• They are not identical, however, because one comes from the biological father and one comes from the biological mother.
• The sex chromosomes, which make up the 23rd pair of chromosomes, determine the sex of a person.
• Human males have an X and Y chromosome (i.e., an X chromosome from the biological mother and a Y chromosome from the biological father); human females have two X chromosomes (i.e., one X chromosome was inherited from each biological parent).
• The much smaller Y chromosome contains genes necessary for the development of male sex.
• In females, only one X chromosome in the female is active in controlling the expression of genetic traits.
• Whether the active X chromosome is derived from the biological mother or biological father is determined within a few days after conception.
• The selection of either X is random for each possible cell line.
• Thus, the tissues of people assigned female at birth normally have on average 50% maternally derived and 50% paternally derived active X chromosomes.

Cell Division

• Two types of cell division occur in humans and many other animals: mitosis and meiosis.
• Mitosis is the process by which DNA is replicated to duplicate somatic cells in the body.
• Each of the two resulting cells should have an identical set of 46 chromosomes, arranged into 23 pairs of chromosomes.
• Meiosis also replicates DNA, but the end product is the formation of gametes or reproductive cells (i.e., ovum and sperm).
• Each cell undergoing meiosis only has a single set of 23 chromosomes.
• Meiosis is divided into two distinct phases, meiosis I and meiosis II.
• As in mitosis, the first step of meiosis I is to replicate the DNA during interphase.
• During metaphase I, all homologous autosomal chromosomes pair up, forming a tetrad of bivalents.
• The X and Y chromosomes are not homologs and do not form bivalents.
• Because the bivalents are lined up, an interchange of chromatid segments can occur in metaphase I. This process is called crossing-over.
• Crossing-over allows for new combinations of genes, increasing genetic variability in the resulting offspring.
• After telophase I, each of the two daughter cells contains one member of each homologous pair of chromosomes and a sex chromosome (23 double-stranded chromosomes).
• During anaphase of meiosis II, the 23 double-stranded chromosomes (two chromatids) divide at their centromeres.
• Each subsequent daughter cell will then receive 23 single-stranded chromatids.
• Meiosis occurs only in the gamete-producing cells found in the testes or ovaries.
• The outcomes are different in males and females.
• In males, meiosis (spermatogenesis) results in four viable daughter cells called spermatids that differentiate into sperm cells.
• In females, gamete formation or oogenesis is quite different.
• After the first meiotic division of a primary oocyte, a secondary oocyte and another structure called a polar body are formed.
• This small polar body contains little cytoplasm, but it may undergo a second meiotic division, resulting in two polar bodies.
• The secondary oocyte undergoes its second meiotic division, producing one mature oocyte and another polar body.
• Four viable sperm cells are produced during spermatogenesis, but only one ovum is produced by oogenesis.

Chromosome Structure

• Cytogenetics is the study of the structure and numeric characteristics of the cell’s chromosomes.
• Chromosome studies can be done on any tissue or cell that grows and divides in culture, but white blood cells or buccal (cheek) samples are frequently used for this purpose.
• After the cells have been cultured, a drug called colchicine is used to arrest mitosis in metaphase so that the chromosomes can be easily seen.
• A chromosome spread is prepared by fixing and spreading the chromosomes on a slide, and they are stained to show banding patterns specific to each chromosome.
• The chromosomes are photographed, and the photomicrographs of each of the chromosomes are cut out and arranged in pairs according to a standard classification system.
• The completed picture is called a karyotype, and the procedure for preparing the picture is called karyotyping.
• In the metaphase spread, each chromosome takes the form of an “X” or “wishbone” pattern.
• The two chromatids are connected by a centromere.
• Human chromosomes are divided into three types according to the position of the centromere.
  • If the centromere is in the center and the arms are of approximately the same length, the chromosome is said to be metacentric.
  • If it is not centered and the arms are of clearly different lengths, it is submetacentric.
  • If it is near one end, it is acrocentric.
• The short arm of the chromosome is designated as “p” for “petite,” and the long arm is designated as “q” for no other reason than it is the next letter of the alphabet.
• The arms of the chromosome are indicated by the chromosome number followed by the p or q designation (e.g., 15p).
• Chromosomes 13, 14, 15, 21, and 22 have small masses of chromatin called satellites attached to their short arms by narrow stalks.
• At the ends of each chromosome are special DNA sequences called telomeres.
• Telomeres allow the end of the DNA molecule to be replicated completely.
• The banding patterns of a chromosome are used in describing the position of a gene on a chromosome.
• Each arm of a chromosome is divided into regions, which are numbered from the centromere outward (e.g., 1, 2).
• The regions are further divided into bands, which are also numbered.
• These numbers are used in designating the position of a gene on a chromosome. For example, Xp22 refers to band 2, region 2 of the short arm (p) of the X chromosome.

Patterns of Inheritance

• The characteristics inherited from a person’s biological parents are carried within genes found along the length of the chromosomes.
• Alternate forms of the same gene are possible, and each may produce a different set of traits or characteristics.

Definitions

• The genotype of a person is the genetic information stored in the sequence of base pairs.
• The phenotype refers to the recognizable traits, physical or biochemical, that are associated with a specific genotype.
• But more than one genotype may have the same phenotype.
• Some brown-eyed people are carriers of the code for blue eyes, and other brown-eyed people are not. Phenotypically, these two types of brown-eyed people appear the same, but genotypically they are different.
• The position of a gene on a chromosome is called its locus, and alternate forms of a gene at the same locus are called alleles.
• When only one pair of genes is involved in the transmission of the phenotype, the term single-gene trait is used.
• Single-gene traits follow Mendelian laws of inheritance.
• Polygenic inheritance involves multiple genes at different loci, with each gene exerting a small effect in determining a trait.
• Multiple pairs of genes, many with alternate alleles, determine most human traits.
• Polygenic traits are predictable to some extent, but with less reliability than single-gene traits.
• Multifactorial inheritance is similar to polygenic inheritance in that multiple alleles at different loci affect the outcome; the difference is that multifactorial inheritance also includes the influence of environmental effects on the phenotype that is produced by these genes.
• Many other types of gene–gene interactions are known.
  • These include epistasis, in which one gene masks the phenotypic effects of another gene.
  • Multiple alleles, in which more than one allele affects the same trait (e.g., ABO blood types).
  • Complementary genes, in which each gene is mutually dependent on the other.
  • Collaborative genes, in which two different genes influencing the same trait interact to produce a phenotype neither gene alone could produce.

Genetic Imprinting

• Certain genes exhibit a “parent of origin” type of transmission in which the parental genomes do not always contribute equally in the development of a person.
• The transmission of this phenomenon is called genetic imprinting.
• Although this phenomenon is somewhat rare, it is influenced by the embryonic environment and remains a topic of high research interest.
• Well-known examples of genomic imprinting are the transmission of the mutations in Prader-Willi and Angelman syndromes.
• Both syndromes result in intellectual disability as a common feature, and both disorders are created by the same deletion mutation in chromosome 15.
• When the deletion is inherited from the biological mother, the infant presents with Angelman syndrome, but when the same deletion is inherited from the biological father, Prader-Willi syndrome results.
• A related chromosomal disorder is uniparental disomy.
• This occurs when two chromosomes of the same number are inherited from one biological parent.
• Normally, this is not a problem except in cases where a chromosome has been imprinted by a biological parent.
• In this case, the offspring will have only one working copy of the chromosome, which can result health problems.

Mendel’s Laws

• A main feature of inheritance is predictability: given certain conditions, the likelihood of the occurrence or recurrence of a specific trait in the offspring is remarkably predictable.
• The units of inheritance are the genes, and the pattern of single-gene transmission can often be predicted using Mendel’s laws of genetic transmission.
• Since Gregor Mendel’s original work was published in 1865, new discoveries have led to some modification of the original laws, but many of the basic principles still hold true.
• Mendel discovered the basic pattern of inheritance by conducting carefully planned experiments with simple garden peas.
• Experimenting with several phenotypic traits in peas, Mendel proposed that inherited traits are transmitted from biological parents to offspring by means of independently inherited factors—now known as genes—and that these factors are transmitted as recessive and dominant traits.
• Mendel labeled dominant factors “A” and recessive factors “a” and then carefully bred generations to track how the traits were inherited in the next generation of plants.
• Geneticists still continue to use capital letters to designate dominant traits and lowercase letters to identify recessive traits.
• The possible combinations that can occur with transmission of single-gene dominant and recessive traits can be described by constructing a figure called a Punnett square using capital and lowercase letters.
• The observable traits of single-gene inheritance are inherited by the offspring from the biological parents.
• The germ cells (i.e., sperm and ovum) of both biological parents undergo meiosis, in which the number of chromosomes is divided in half (from 46 to 23) and each germ cell receives only one allele from each pair. This fact was described in Mendel’s first law.
• According to Mendel’s second law, the alleles from the different gene loci segregate independently and recombine randomly in the offspring.
• People in whom the two alleles of a given pair are the same (AA or aa) are called homozygotes.
• Heterozygotes have different alleles (Aa) at a gene locus.
• A recessive trait is one that appears in the phenotype only in a homozygous (aa) pairing; a dominant trait is one that appears in the phenotype in either a homozygous (AA) or a heterozygous (Aa) pairing.
• If the trait follows simple Mendelian inheritance, then all people with a dominant allele in either one or two copies will show the phenotype for that trait.
• For example, the genes for blond hair are recessive and those for brown hair are dominant. Therefore, only people with a genotype having two alleles for blond hair would be blond; people with either one or two brown alleles would have brown hair.
• Sometimes, the person that is heterozygous for a recessive trait (Aa) is called a carrier. That individual will not exhibit the phenotype for the recessive trait, but they “carry” the allele for the recessive trait. If they have offspring with someone that exhibits and is homozygous for the recessive trait, or with someone who is also a carrier, then that offspring could also exhibit the recessive trait. Such a situation might occur when two people that have a phenotype of brown eyes have a child with blue eyes. Brown (B) eyes are dominant to blue eyes (b), so both biological parents had to be genotypically heterozygous (Bb).

Pedigree

• A pedigree is a graphic method for portraying a family history for an inherited trait.
• It is constructed from a carefully obtained family history and is useful for tracing the pattern of inheritance for a particular trait.

Gene Technology

• The past several decades have seen phenomenal advances in the field of genetics.
• These advances have included the completion of the Human Genome Project, the establishment of the International HapMap Project to map the haplotypes of variations in the human genome, and the development of methods for applying the technology of these projects to the diagnosis and treatment of disease.
• Many healthcare professions also have established clinical competencies for their specific professions regarding genetics, as the application of genetic technology is progressively becoming more evident in all areas of disease screening and management.
• While genetic counselors may lead the effort, all healthcare professionals need to be able to answer questions about testing and how this knowledge may or may not influence the course of one’s health.

Genetic Mapping

• Genetic mapping is the assignment of genes to a specific locus or to specific parts of the chromosome.
• Another type of mapping strategy, the haplotype map, focuses on identifying the slight variations in the human genome that affect a person’s susceptibility to disease and responses to environmental factors such as microbes, toxins, and drugs.

The Human Genome Project

• The Human Genome Project, initiated in 1990 and completed in 2003, sought to identify all the genes in the human genome.
• The international project was charged with both determining the precise locations of genes and also exploring technologies that would enable the sequencing of large amounts of DNA with high accuracy and low cost.
• Although we now take the availability of the human genome sequence for granted, some of what was discovered was quite unexpected, and much still remains to be known.
• One initial revelation of the project was that humans have far fewer genes than the 100,000 that were initially predicted from the number of different proteins in our body.
• Another surprising finding that came from the Human Genome Project was mentioned earlier in this chapter: On average, any two unrelated people will still share 99.9% of their DNA sequence, indicating that the remarkable diversity among people is carried in about 0.1% of our DNA.

Genetic Mapping Methods

• Many methods have been used for developing genetic maps. The most important ones are family linkage studies, gene dosage methods, and hybridization studies.
• Often, the specific assignment of a gene locus is made using information from several mapping techniques.

Linkage Studies

• Linkage studies assume that genes occur in a linear array along the chromosomes.
• During meiosis, the paired chromosomes of the germ cells sometimes exchange genetic material because of crossing-over.
• This exchange usually involves more than one gene; large blocks of genes (representing large portions of the chromosome) are often exchanged.
• The closer together two genes are physically on the same chromosome, the greater the chance is that they will be passed on together to the offspring.
• When two inherited traits occur together at a rate that is greater than would occur by chance alone, they are said to be linked.
• Linkage analysis can sometimes be used clinically to identify affected people in a family with a known genetic defect.

Hybridization Studies

• When two somatic cells from different species are grown together in the same culture, occasionally they fuse to form a new hybrid cell.

Somatic cell hybridization

• Somatic cell hybridization involves the fusion of human somatic cells with those of a different species (typically, the mouse) to yield a cell containing the chromosomes of both species.
• Because these hybrid cells are unstable, they begin to lose chromosomes of both species during subsequent cell divisions.
• This makes it possible to obtain cells with different partial combinations of human chromosomes.
• The proteins of these cells are then studied with the understanding that for a protein to be produced, a certain chromosome must be present and, therefore, the coding for that protein must be located on that chromosome.

In situ hybridization

• In situ hybridization involves the use of specific sequences of DNA or RNA to locate genes.
• Both DNA and RNA can be chemically tagged with radioactive or fluorescent markers.
• These chemically tagged DNA or RNA sequences are used as probes to detect a gene location or the presence of a specific variant.
• If the probe matches the complementary DNA of a chromosome segment, it hybridizes and remains at the precise location (therefore the term in situ) on a chromosome.
• By tracking the radioactive or fluorescent markers, the location of the probe binding can then be found.

Haplotype Mapping

• As work on the Human Genome Project progressed, many researchers reasoned that identifying the common patterns of DNA sequence variations in the human genome would be possible.
• An international project, known as the International HapMap Project, was organized with the intent of developing a haplotype map of these variations.
• Sites in the DNA sequence where people differ at a single DNA base are called single nucleotide polymorphisms (SNPs, pronounced “snips”).
• A haplotype consists of the many closely linked SNPs on a single chromosome that generally are passed as a block from one generation to another in a particular population.
• One of the motivating factors behind the HapMap Project was the realization that the identification of a few SNPs was enough to uniquely identify the haplotypes in a block.
• The specific SNPs that identify the haplotypes are called tagging SNPs.
• This approach reduces the number of SNPs required to examine an entire genome and makes genome scanning methods much more efficient in finding regions with genes that contribute to disease development.
• Much attention has focused on the use of SNPs indicating disease susceptibility in one population vs. another, as well as determining appropriate medications and therapies based upon genotype.

Recombinant DNA Technology

• The term recombinant DNA refers to a combination of DNA molecules that are not found together in nature.
• Recombinant DNA technology makes it possible to identify the DNA sequence in a gene and produce the protein product encoded by a gene.
• The specific nucleotide sequence of a DNA fragment can often be identified by analyzing the amino acid sequence and mRNA codon of its protein product.
• Short sequences of base pairs can be synthesized, radioactively labeled, and subsequently used to identify their complementary sequence.
• In this way, identifying both normal and abnormal gene structures is possible.

Gene Isolation and Cloning

• The gene isolation and cloning methods used in recombinant DNA technology rely on the fact that the genes of all organisms, from bacteria through mammals, are based on a similar molecular organization.

• Gene cloning requires cutting a DNA molecule apart, modifying and reassembling its fragments, and producing copies of the modified DNA, its mRNA, and its gene product.

• The DNA molecule is cut apart by using a bacterial enzyme, called a restriction enzyme, that binds to DNA wherever a particular short sequence of base pairs is found and cleaves the molecule at a specific nucleotide site.

• In this way, a long DNA molecule can be broken down into smaller, discrete fragments, one of which contains the gene of interest. Many restriction enzymes are commercially available that cut DNA at different recognition sites.

• The fragments of DNA can then often be replicated through insertion into a unicellular organism, such as a bacterium.

• To do this, a cloning vector such as a bacterial virus or a small DNA circle that is found in most bacteria, called a plasmid, is used.

• Viral and plasmid vectors replicate autonomously in the host bacterial cell.

• During gene cloning, a bacterial vector and the DNA fragment are mixed and joined by a special enzyme called a DNA ligase.

• The recombinant vectors formed are then introduced into a suitable culture of bacteria, and the bacteria are allowed to replicate and express the recombinant vector gene.

• While this method is a staple of many geneticists, the methods of recombinant DNA technology can also be used in the development of pharmaceuticals. For example, recombinant DNA technology is used in the manufacture of human insulin that is used to treat diabetes mellitus.

DNA Fingerprinting

• The technique of DNA fingerprinting also uses recombinant DNA technology, as well as basic principles of medical genetics.
• Using restriction enzymes, DNA is first cleaved at specific regions.
• The DNA fragments are separated according to size by electrophoresis and denatured (by heating or treating chemically) so that all the DNA is single stranded.
• The single-stranded DNA is then transferred to nitrocellulose paper, baked to attach the DNA to the paper, and treated with series of radioactive probes.
• After the radioactive probes have been allowed to bond with the denatured DNA, radiography is used to reveal the labeled DNA fragments.
• When used in forensic pathology, this procedure is applied to specimens from the suspect and the forensic specimen.
• This can be done with even very small samples of DNA (a single hair or a drop of blood or saliva) using amplification by polymerase chain reaction.
• The DNA banding patterns between samples are analyzed to see if they match.
• With conventional methods of analysis of blood and serum enzymes, a 1 in 100 to 1,000 chance exists that the two specimens match because of chance.
• With DNA fingerprinting, these odds are 1 in 100,000 to 1 million.

Gene Therapy

• Although quite different from inserting genetic material into a unicellular organism such as bacteria, techniques are available for inserting genes into the genome of intact multicellular plants and animals.
• Promising delivery vehicles for these genes are the adenoviruses.
• These viruses are ideal vehicles because their DNA does not become integrated into the host genome.
• However, repeated inoculations are often needed because the body’s immune system usually targets cells expressing adenovirus proteins.
• This type of therapy remains one of the more promising methods for the treatment of genetic disorders such as cystic fibrosis, certain cancers, and many infectious diseases.
• Two main approaches are used in gene therapy: transferred genes can replace defective genes, or they can selectively inhibit the expression of deleterious genes.
• Cloned DNA sequences are usually the compounds used in gene therapy.
• However, the introduction of the cloned gene into the multicellular organism can influence only the few cells that get the gene.
• An answer to this problem would be the insertion of the gene into a sperm or ovum; after fertilization, the gene would be replicated in all of the differentiating cell types.
• Another potential therapeutic has been found in the advent of CRISPR-Cas9 technology