Midterm Lesson 1: Mendelian Genetics PDF

Midterm Lesson 1: Mendelian Genetics - In honor of Gregor Johann Mendel who experimented with pea plants Single-Gene Inheritance - AKA Mendelian Inheritance - Inheritance of a trait or characteristics controlled by a single gene - Simplest form of inheritance - Ex: dimples,...

Midterm Lesson 1: Mendelian Genetics - In honor of Gregor Johann Mendel who experimented with pea plants Single-Gene Inheritance - AKA Mendelian Inheritance - Inheritance of a trait or characteristics controlled by a single gene - Simplest form of inheritance - Ex: dimples, widow’s peak, earlobes, albinism Single-Gene Traits - AKA monofactorial traits/Mendelian Characteristics: 1. Relationship with the affected relative 2. Tests can sometimes predict the risk of developing symptoms 3. More common in some populations than others 4. It may be “fixable” Examples: Widow’s peak, dimples, earlobes, albinism Eye Color - Color of the iris Due to melanin pigments, which come in two forms–the dark brown/black eumelanin, and the red-yellow pheomelanin - Melanocytes Cells that produce melanin, which is stored in structures called melanosomes in the outermost layer of the iris People differ in the amount of melanin and number of melanosomes, but have about the same number of melanocytes in their eyes - Nuanced of eye color Light vs dark brown, clear blue vs greenish or hazel Arise from the distinctive peaks and valleys at the back of the iris ❖ Thicker regions darken appearance of the pigments, rendering brown eyes nearly black in some parts and blue eyes closer to purple ❖ The bluest eyes have thin irises with very little pigment - Single gene on chromosome 15, OCA2 Confers eye color by controlling melanin synthesis If missing, albinism results, causing pale skin and red eyes Recessive allele of this gene confers blue color and a dominant allele confers brown - HERC2 Near the OCA2 gene on chromosome 15 Controls expression of the OCA2 gene Recessive allele abolishes the control over OCA2, and blue eyes result A person must inherit two copies of the recessive allele in HERC2 to have blue eyes Found in many species, indicating that it is ancient and important, because it has persisted Definition of Terms Allele - Alternative form of gene (unit of inheritance) and present in two copies  1. Dominant Allele ➔ One allele that masks another, has an effect even when present in just one copy ➔ Denoted by a capital letter; ex: W (widow’s peak/dominant) 2. Recessive Allele ➔ Masked, must be present on both chromosomes of a paired allele to be expressed ➔ Denoted by a small letter; ex: w (straight hairline/recessive) Homozygous - Two identical alleles (true-breeding) - Ex: WW, ww Heterozygous - Two different alleles (non-true breeding/hybrid) - Ex: Ww Compound Heterozygote - Individual with two different recessive alleles for the same gene Genotype - Underlying genetic instructions - Describes the organism’s alleles - Paired alleles present Phenotype - Visible trait, outward expression of an allele combination 1. Dominant Allele/Trait ➔ One trait masking another 2. Recessive Allele ➔ Masked trait 3. Wild Phenotype ➔ Most common expression of a particular allele combination in a population ➔ May be recessive or dominant ➔ Ex: Skin Color (caucasoid, mongoloid, and negroid); Tigers (orange fur and black stripes) 4. Mutant Phenotype ➔ Variant of a gene’s expression that arises when the gene undergoes a change (mutation) ➔ Ex: albinism; Tigers (white tiger with dark stripes and albino tigers) - NOTE: An organism’s appearance does not always reveal its alleles Mendel’s Experiments 1857-1863 - Mendel crossed and cataloged traits in 24,034 plants through several generations - Termed units of heredity as “elementen” which was renamed by William Bateson (1909) to “genes” which is a Greek word for “give birth to” - He deduced that consistent ratios of traits in the offspring indicated that the plants transmitted distinct units, and his two hypotheses proposed how this happens 1866 - Mendel published “Experiments on Plant Hybridization” 1901 - Hugu de Vries (Holland), Carl Correns (Germany), and Erich von Ischermak (Austria) who all worked independently, used Mendel’s theory making him known into the field of genetics Why Pea Plants? Easy to grow Develop quickly Have many traits that take one of the easily distinguishable forms Analyzing Genetic Crosses Parental Generation (P1) - First generation First Filial Generation (F1) - Second generation Second Filial Generation (F2) - Next generation Punnett Square - Named after Reginald C. Punnett who first devised this approach - Used to predict possible genotypes and phenotypes of offspring resulting from a cross between two individuals - Represents how genes in gametes join if they are on different chromosomes - In humans, females genotype should be written on the horizontal part, while the male is on the vertical part - One allele in a sex cell  When Mendel crossed short plants (tt) with true-breeding tall plants (TT), the seed grew into F1 plants that were all tall (genotype Tt) Mendel self-crossed the F1 plants and the progeny were TT, tt, and Tt ❖ A TT individual resulted when a T sperm fertilized a T oocyte ❖ A tt plant resulted when a t oocyte met a t sperm ❖ A Tt individual resulted when either a t sperm fertilized a T oocyte, or a T sperm fertilized a t oocyte Because two of the four possible gamete combinations produce a heterozygote, and each of the others produces a homozygote, the genotypic ratio expected of a monohybrid cross is 1TT:2Tt:1tt The corresponding phenotypic ratio is three tall plants to one short plant, a 3:1 ratio Mendel’s Observations True-breeding - Producing the same phenotype - Same trait as parent - Homozygous dominant or recessive - Ex: A true-breeding purple plant crossed with itself will always produce offspring that are purple as well Monohybrid Cross - Follows one trait - Self-crossed plants are hybrids - True-breeding plants with two forms of a single trait are crossed - Formed by one homozygous dominant and one homozygous recessive - Hybrids hide one expression of a trait which reappears when hybrids are self-crossed Mendel’s explanation of this phenomenon: Gametes distribute “elementen” because these cells physically link generations Paired sets of elementen separate as gametes form When gametes join at fertilization, the elementen combine anew ❖ Law of Segregation ➔ Elementens are segregated in each sex cells ➔ Distribution of alleles of a gene into separate gametes during meiosis - Mendel conducted up to 70 hybrid self-crosses for each of the seven traits  Test Cross (Backcross) - Crossing an individual of unknown genotype with a homozygous recessive individual ❖ The only genotype than can be identified by its phenotype–that is, a short plant is always tt ❖ A “known” that can reveal the unknown genotype of another individual to which it is crossed - Determine the genotype of an individual showing a dominant phenotype - The parent is the one being identified - Ex: Tall pea plant (dominant), is it homozygous or heterozygous? - Selection of an organism: Displaying the dominant phenotype 1. Selection of the test cross parent: Homozygous recessive (ALWAYS) 2. Observation 3. Interpretation of results ❖ Ex: Curly hair (dominant); straight hair (recessive) Mode of Inheritance - Pattern by which a genetic trait or disorder is passed from one generation to next within a family - A way that a trait or disorder is passed depends on whether a. The gene that determines it is on an autosome or sex chromosome b. Whether the allele is recessive or dominant - Includes 1. Autosomal Dominant Inheritance ➔ Trait or disorder is caused by a dominant allele located on one of the autosomal chromosomes  ➔ A single copy of dominant allele will exhibit the trait or disorder ➔ Criteria: 01. Male and female can be affected. Male to male transmission can occur. 02. Male and female transmit the trait with equal frequency 03. Successive generations are affected 04. Transmission stops after a generation in which one inherits the mutation stops producing offspring 05. Affected individual has an affected parent, unless he or she has a de novo mutation ➔ Many affected individuals are heterozygous ➔ Homozygous dominant phenotype is either lethal or very rare Ex: Neurofibromatosis, Huntington Disease 2. Autosomal Recessive Inheritance ➔ Caused by a recessive allele located on one of the autosomal chromosomes ➔ Two copies of the recessive allele (homozygous) is needed to exhibit trait or disorder ➔ Affected individuals Homozygous recessive ➔ Carriers of the trait or disorder Heterozygous and asymptomatic ➔ Criteria: 01. Male and female can be affected 02. The affected individual can transmit the gene, unless it causes death before reproductive age 03. Affected individuals have a homozygous recessive genotype, whereas in heterozygotes (carriers) the wild type allele masks expression of mutant allele 04. Trait or disorder can skip generations 05. Parents of affected individual are heterozygous or have the trait ➔ Ex: Albinism and cystic fibrosis 3. Sex-Linked Inheritance ➔ Pattern of inheritance of genetic traits or disorders that are located on the sex chromosomes a. Y-linked Inheritance ➔ Genes on Y chromosome and traits or disorders are rare because it has few genes ➔ Male-to-male (father to son) ➔ No affected females ➔ Ex: Male infertility, congenital adrenal hypoplasia, RP2 b. X-linked Dominant Inheritance ➔ Dominant allele located on X chromosome ➔ Expressed in females in one copy and are more likely to be affected ➔ Much more severe effects in males ➔ Passed from male to all daughters, but not to sons ➔ Ex: Rett Syndrome, incontinentia pigmenti, congenital hypertrichosis/werewolf syndrome c. X-linked Recessive Inheritance ➔ Recessive allele is located on the X chromosome  ➔ Always expressed in males (inherited from heterozygous or homozygous mother) ➔ Expressed in females if causative allele is present in two copies (from affected father and affect heterozygous mother) ➔ Females are usually carriers (heterozygotes and asymptomatic) ➔ Ex: Color blindness, hemophilia and ichthyosis Solving a Problem in Following a Single Gene 1. List all possible genotypes and phenotypes for the trait 2. Determine the genotypes of the individuals in the first (P1) generation. Use information about those individuals’ parents. 3. After deducing genotypes, derive the possible alleles in gametes each individual produces. 4. Unite these gametes in all combinations to reveal all possible genotypes. Calculate ratios for the F1 generation. 5. To extend predictions to the F2 generation, use the genotypes of the specified F1 individuals and repeat steps 3 and 4. Inheritance of More Than One Gene Law of Independent Assortment - Mendel’s Second Law of Inheritance: For two genes on different chromosomes, the inheritance of one gene does not influence the chance of inheriting the other gene - The two genes are said to “independently assort” because they are packaged into gametes at a random - Happens in meiosis; RRYY x rryy Solving a Problem in Following Multiple Genes - A Punnett square for three greens has 64 boxes; for four genes 256 boxes - Probability An easier way to predict genotypes and phenotypes in multigene crosses Basis of Punnett squares Predicts the likelihood of an event - Application of Probability Theory Can predict the chance that parents with known genotypes can produce offspring of a particular genotype - Product Rule States that the chance that two independent event swill both occur equals the product of the chances that either event will occur alone ➔ Figure 4.11 shows the probability of obtaining a plant with wrinkled, green peas (genotype rryy) from dihybrid (RrYy) parents ➔ A Punnett square depicting a cross of two Rr plants indicates that the probability of producing rr progeny is 25% or ¼ ➔ The chance of two Yy plants producing a yy plant is ¼ ➔ Therefore, the chance of dihybrid parents (RrYy) producing homozygous recessive (rryy) offspring is ¼ multiplied by ¼ or 1/16 Dihybrid Cross - Two genes and traits are followed Mendel looked at the: a. Seed Shape: either round or wrinkled (determined by the R gene) b. Seed Color: either yellow or green (determined by the Y gene) c. Punnett Square d. Write the genotypes of the offspring in each box and determine how many of each phenotype you have - iFOIL method sa bago mag start sa Punnett square Pedigree Analysis  - Chart that display family relationships and depict which relatives have specific phenotypes and sometimes genotypes - May also include molecular data, test results, and information on variants of multiple genes - Pedigree in Genetics Indicates inherited diseases or traits as well as relationships and ancestry - Lines link shapes Vertical Lines: represent generations Horizontal Lines: connect two shapes at their centers depict partners Shapes connected by vertical lines that are joined horizontally: siblings Squares: males Circles: females Diamonds: individuals of unspecified sex Roman Numerals: generations Arabic numeral or names: individuals Pedigree - The earliest ones were strictly genealogical (tracing lines of family descent), not indicating traits - Arose in the 15th century, from the French pie de grue, which means “crane’s foot” - Pedigrees Now  Important both for helping families identify the risk of transmitting an inherited illness and as starting points for identifying and describing, or annotating, a gene from the human genome sequence - Can be inconclusive if a dominant trait or illness does not impair fertility - An individual with a de novo dominant mutation or two autosomal recessive alleles inherited from carrier parents can appear the same in a pedigree - Pedigrees and Punnett squares Can predict the phenotypic and genotypic classes of offspring in cases of conditional probability in which knowing a parent’s genotype restricts the possible genotypic classes of an offspring - Comparing exome or genome sequences in parents-child trios when the child has an unrecognized syndrome and the parents are unaffected can distinguish autosomal recessive inheritance from the parents from a de novo dominant mutation Midterm Lesson 2: Modification of Mendelian Ratios - Phenomena that seem like exceptions of the Mendelian laws but are not Lethal Allele Combinations Lethal Genotypes - Genotype (allele combination) that causes death before reproduction is possible; homozygous - Precludes reproduction - Ex: Achondroplastic dwarfism–autosomal dominant disease that is most often the result of spontaneous or new mutation ❖ Impaired bone growth particularly in the limbs ❖ Disproportionate limbs and large head compared to body ❖ Heterozygous shows the trait/disorder ❖ Problem: If two people with achondroplasia have children, what will be the probability of inheriting achondroplasia and of having a normal height? Multiple Alleles A person has two alleles for any autosomal gene which is one part of each homologous chromosome but a gene can also exist in more than two allelic forms in a population because it can mutate Three or more alleles of the same gene are involved Different allele combinations can produce variations in the phenotype - More alleles, more variations in the phenotype Compound Heterozygote - Individual with two different mutant alleles for the same gene Simplest case: 3 alternative alleles of one gene exist - Ex: ABO blood group system in humans  ❖ Involves 3 alleles: IA (I: isoagglutinogen), IB, and IO ❖ Presence or absence of specific antigens in the surface of RBCs ABO Blood Group System Discovered by Karl Landsteiner Characterized by the presence or absence of antigens (A&B) on the surface of RBCs When individuals are tested using antisera (determine what antigen is present in the blood cells of an individual) that contain antibodies against the A or B antigen, 4 phenotypes are revealed, either: ❖ The A antigen (A phenotype) ❖ The B antigen (B phenotype) ❖ The A and B antigens (AB phenotype) ❖ Or neither antigen (O phenotype) When blood is tested using the Anti-A and Anti-B reagents, the presence of any of the antigen will cause the antibodies to bind with the RBCs ❖ Cause agglutination: happens when there is a clumping RBCs Antibody: specific type of immune system proteins, called immunoglobulins; present in plasma/serum; naturally occurring antibodies Antigen: molecule, usually protein, capable of initiating the formation of antibodies; present in surface of RBCs ABO blood group system in humans: involves 3 alleles: IA, IB, and IO - The I designation stands for isoagglutinogen (or isoantigen) - If there is clumping, agglutination occurs - IA and IB are codominant (joint expression of both alleles in a heterozygote) Different Dominance Relationships 1. Complete Dominance One allele is expressed while the other is not Dominant allele completely masks the recessive allele Ex: Mendel’s pea plants 2. Incomplete Dominance Partial dominance Neither allele dominates the other Heterozygous phenotype is an intermediate or blended expression of the two alleles 3 genotypes = 3 phenotypes Ex: flower of snapdragon In humans: Tay-Sachs Disease Absence or dysfunction of the beta-hexosaminidase A enzyme Homozygous dominant: full enzyme level Heterozygotes: intermediate level of enzyme Homozygous recessive: no enzyme  Autosomal recessive inheritance 3. Codominance Different alleles are both expressed in a heterozygote Neither allele is dominant over the other and both contribute to the observable characteristics of the organism Both allele have a contribution to the phenotype Ex: ABO blood group Sample Problem: ABO Blood Group System What are the possible blood types of the children whose mother has blood type A and father with blood type B? Epistasis - Effect of one gene masks or modifies the expression of another gene - Ex: hh blood group (Bombay phenotype) = interaction between H gene and the I gene (confers ABO blood type) H antigen: building block for the production of the antigens within the ABO blood group HH and Hh: has or forms the H antigen hh individuals cannot produce antigens of the A and B, and appear to be O-type Sample Problem: ABHh x ABHh What are the frequencies of the apparent blood groups among the offspring? Penetrance All-or-none expression of a genotype Frequency of expression of disease phenotype in individuals with a gene lesion Described numerically Statistically calculated from populations whose genotype we know Two Types a. Complete Penetrance: Gene (s) for a trait are expressed in all the population who have the genes ❖ Allele combination produces phenotype to all individuals who have the gene b. Incomplete Penetrance: Genetic trait is expressed only in part of the population ❖ Not all individuals will express the phenotype Expressivity Severity or extent Range of phenotypes in individuals with the same gene lesion Variable Expressivity: symptoms varies in intensity among different people Ex: polydactyly Pleiotropy A single gene influences or controls multiple seemingly unrelated traits or phenotypic characteristics A single gene has multiple effects on the phenotype of an organism Difficult to trace through families because people with different subsets of symptoms may appear to have different disorders Molecularly, happens if one protein affects different body parts, or participates in more than one biochemical reaction, or has different effects in different amounts Ex: porphyria variegata ❖ Autosomal dominant disease ❖ Common in royal families ❖ Present after puberty ❖ Caused by a single gene mutation which has an effect on the production of the protoporphyrinogen oxidase enzyme ➔ Has a crucial role in the heme synthesis pathway which is an important component of hemoglobin ➔ Leads to accumulation of porphyrin which can results to various symptoms and complications Genetic Heterogeneity Mutations in different genes that produce the same phenotype Multiple genetic alterations can lead to a common outcome or trait Highlight the existence of genetic diversity underlying a particular phenotype Ex: blindness ❖ Mutations in more than 100 genes cause degeneration of the retina Phenocopy An acquired condition that resembles a genetic condition or disease Phenotypes caused by environmental exposures, infections, nutritional deficiencies, medications, or other non-genetic factors Acquired condition that resembles a genetic condition or disease Highlight the potentials of environmental or non-genetic influences to produce a phenotype that resembles a genetic disorder ❖ Emphasizes the need for a careful evaluation and consideration of those factors when diagnosing a condition or interpreting a genetic test results Ex: Limb birth defect ❖ Caused by the drug thalidomide is a phenocopy of the inherited illness phocomelia Mitochondrial Genes Maternally inherited ❖ Only passed from an individual’s mother ❖ Does not penetrate the egg cell, only the sperm head Sperm head does not include mitochondria Mitochondrion is found in the midsection ❖ Responsible in giving energy for the movement of the sperm tail When mitochondria from sperm enters an oocyte ❖ They are usually selectively destroyed early in the development Pedigree for inheritance of mitochondrial genes Features of mtDNA Circular, 16,569 base pairs, with 37 genes (24 encode RNA molecules and 13 encode proteins that function in cellular respiration) Mutates faster than nuclear DNA because it has fewer types of DNA repair  Inherited from the mother only No DNA-associated proteins (histones) mt genes are not interrupted by DNA sequences that do not encode protein Many copies per mitochondrion and per cell Mitochondria with different alleles for the same gene can reside in the same cell Mitochondrial Diseases Mitochondrial myopathies ➔ Occurs in muscle tissues ➔ Disruption in the normal structure and distribution of mt within the muscle fibers Skeletal muscle ➔ Affected tissues are those normally with many mitochondria May also result from mutations in nuclear genes that are essential for mitochondrial function Hallmark: red-ragged fibers ❖ Refer to the muscle fibers that display an abnormal accumulation of mitochondria ❖ Mitochondria is often enlarged and has an abnormal shape Heteroplasmy ➔ Mutated mitochondria and normal mitochondria in the same cell Theoretically, a woman with mt disease can avoid passing down the trait if the mt is replaced with a healthy mt from a donor mtDNA Reveals the Past Forensics ➔ To link suspects to crimes Can identify war dead and support or challenge historical records Often better preserved in ancient remainings compared to the DNA found in the nucleus Midterm Lesson 3: Nucleic Acids: DNA and RNA Nucleic Acids Molecular repositories (location) for genetic information and are jointly referred to as the ‘molecules of heredity’ Biomolecules which has a very important role in storing, transmitting, and expressing genetic information 1. Deoxyribonucleic Acid (DNA) - Serves as the genetic material in all living organisms as well as in most viruses - Main job: Serve as a medium for long term storage and transmission of genetic information 2. Ribonucleic Acid - Involved in protein synthesis and sometimes in the transmission of genetic information  - Main job: Transfer genetic code needed for the creation of proteins, particularly proteins from the nucleus to the ribosomes Discovery of Nucleic Acids 1869 (Friedrich Miescher) - Isolated nuclei from pus cells (WBCs) and found that they contained “nuclein” - Upon his observations, nuclein is a phosphate-rich substance which was quite different from the carbohydrates, proteins, and fats 1880s (Emil Fischer) - Discovered the component of nucleic acids: purine and pyrimidines 1894 (Geheimrat Albrecht Kossel) - Recognizes that nucleins are associated with histones - He also found out that these histones were basic proteins and - 1910: Offered a nobel prize for physiology or medicine for demonstrating the presence of two purines and two pyrimidine bases in nucleic acids 1899 (Richard Altmann) - It was found out that the nuclein has acid properties - Introduced the term “nucleic acid” 1909 (P.A./Phoebus Aaron Theodor Levene) - Recognized the 5-carbon ribose sugar and later discovered deoxyribose in nucleic acids 1914 (Robert Feulgen) - Demonstrated a color test known as Feulgen test for the DNA - Used a staining technique selective for the DNA 1929 (P.A. Levene) - Stressed that there are two types of nucleic acids: DNA and RNA - Also discovered that the nucleic acid has three parts: 1. Sugar 2. Nitrogen-containing base 3. Phosphorus-containing component - Observed that those three parts are present in equal proportions ❖ He then deduced that a nucleic acid building block must contain one of each component 1950s (Erwin Chargaff) - Discovered that several species contain equal amounts of the bases: ❖ A = T; and G = C (Chargaff’s rule) - Used paper chromatography and UV spectroscopy to examine the abundance of bases in DNA of different species ❖ Upon checking, he noticed something very odd ❖ The DNA of species contains equal amounts of bases (adenine, thymine, guanine, and cytosine) ➔ Hinted the base pair makeup of DNA ❖ Shared his discovery with Watson and Francis Crick in1952 1950s (Maurice Wilkins and Rosalind Franklin) - Bombarded DNA with X-rays (X-ray diffraction), then deduced the overall structure of the molecule from the patterns in which the X-rays were deflected - Upon observation, particularly by Franklin, she noticed that there are two forms of DNA: a. A Form: somehow dry and is also crystalline; prevalent under high salt or dehydration conditions b. B Form: wetter type; present under aqueous/low salt conditions; exist in cells - Franklin took many pictures of the B form and called it the Photo 51  - Franklin was not able to published her findings - Wilkins took the Photo 51 from Franklin’s desk and gave it to Francis Crick and James Watson 1953 (Linus Pauling) - Suggested a triple helix structure of DNA in which the phosphate structure and sugar are inside and the bases are on the outside ❖ This misinterpretation is due to the mixture of A and B form of DNA that Pauling saw 1953 (James Watson and Francis Crick) - Constructed the double-helical model for DNA - Published their findings in April 25, 1953 issue of Nature magazine - Based their model of the Photo 51 by Rosalind Franklin - Received a Nobel Prize Nucleic Acid: Structure DNA Structure - Double-stranded molecule with a long chain of nucleotides 1. Nucleotide - Single building block of DNA - Deoxyribose sugar, phosphate group, and nitrogenous base Nitrogenous Bases - Information-containing parts of DNA ❖ Form sequences - DNA sequences are measured in numbers of base pairs Two Classifications: - Purines: Adenine (A) and Guanine (G) ❖ Two-ring structure - Pyrimidines: Cytosine (C) and Thymine (T) ❖ Single ring structure Sugar-phosphate Backbone - Formed when the nucleotides are joined into long chains when strong attachments called phosphodiester bonds form between the deoxyribose sugars and the phosphates - Important because it forms the structural framework of nucleic acids - Plays a crucial role in nucleic acids stability and function - Antiparallelism: Opposing orientation of the two nucleotide chains in a DNA; the sugar-phosphate backbone have different directions ❖ Becomes apparent if carbons in the sugar are numbered ❖ The 5’ and the 3’ carbons establish the directionality of each DNA strands - The DNA has a specific purine and pyrimidine couples ❖ In DNA, these bases are partnered ❖ Adenine is paired up always with Thymine  ❖ Guanine is paired up always with Cytosine ❖ The pairing of these bases are joined together by chemical attractions called hydrogen bonds ➔ Thymine and Adenine are joined together by two hydrogen bonds ➔ Cytosine and Guanine are joined together by three hydrogen bonds Complementary Base Pairs Example 1: Given the DNA strand = ACGTCG, what is its complementary strand? TGCAGC Example 2: Given the DNA strand = CATGCATT, what is its complementary strand? GTACGTAA DNA Configuration in the Nucleus DNA molecules are extremely long - DNA of smallest chromosome: 14 mm long if stretched out, but is packaged into a chromosome (um long) DNA Packaging - Process by which the long, linear DNA molecules are tightly compacted and organized into a more condensed structure within the cell 1. Naked DNA wrapped around special proteins called histones 2. The combination of DNA and histones is the nucleosomes which are packaged into a thread resembling beads on a string 3. The linker DNA tighten the nucleosomes to become fibers which are about 30 nm in diameter 4. End result: chromatin fiber which is not only made up of DNA but also of 30% histone, 30% DNA scaffold and other DNA-binding proteins , 10% RNA Remaining 30% is the DNA component of the chromatin 5. Chromatin will be coiled forming the chromatin loops which are then compacted together and coiled leading to the formation of chromosome RNA Structure - Single-stranded molecule in most of its biological roles and has a shorter chain of nucleotides Nucleotides - Ribose sugar, phosphate group, and nitrogenous base Nitrogenous Bases - Purines: Adenine (A) and Guanine (G) - Pyrimidines: Cytosine (C) and Uracil (U) ❖ Lacking a methyl group on its ring DNA vs RNA DNA RNA Usually double-stranded Usually single-stranded Thymine as base Uracil as base Deoxyribose as the sugar Ribose as the sugar Maintains protein-encoding informations Carries protein-encoding information and controls how information is used Cannot function as an enzyme Can function as an enzyme Persists Transient (short-lasting) DNA Replication - Process by which DNA makes a copy of itself during cell division - Usually happens in the S-phase of the interphase Central Dogma - Where DNA replication is a part of - Genetic flow of information from the DNA to RNA to protein Parts: 1. DNA Replication - DNA makes a copy of itself 2. Transcription - The information contained in the DNA is converted to RNA 3. Translation - The information contained in RNA is converted to protein Genetic Material - Must carry out two jobs according to Francis Crick: 1. Duplicate itself 2. Control the development of the rest of the cell in a specific way  - The only molecule that fulfills this important requirement is the DNA Discovery of the Genetic Material 1869 (Friedrich Miescher) - Isolated the genetic material from WBC nuclei; noted it had an acidic nature containing Nitrogen and Phosphorus and called it nuclein 1902 (Archibald Garrod) - First to link inherited disease and protein - People who had certain inborn errors of metabolism did not have certain enzymes 1928 (Frederick Griffith) - Investigated virulence in Diplococcus (now known as Streptococcus pneumoniae) ❖ Ability of microorganism to cause severe disease or infection in a host or itself - 2 strains = rough type (avirulent) and smooth type (virulent) - Use two control setups for his experiment: ❖ Rough nonvirulent (type R): healthy mouse ❖ Smooth virulent type (type S): mouse dies - Experimental variable ❖ Heat-killed smooth virulent ❖ Transform Principle: The type S bacteria transferred its killing trait to the type R bacteria 1944 (Oswald T. Avery, Colin Macleod, and M.J. MacCarty) - Identified the transforming material as DNA by preparing boiled virulent bacterial cell lysates and sequentially treated them with enzymes ❖ Prepared by breaking open the bacterial cells and releasing its contents and sequentially treated it with enzymes: Protease and DNase ❖ Protease: capable of breaking down proteins only; only DNA left in the lysates ❖ DNase (Deoxyribonuclease): capable of breaking down the DNA; protein is the only left in the lysates - Conclusion: “transforming principle” in Griffith’s experiment was DNA 1953 (Alfred Hershey and Martha Chase) - Confirmed that the DNA of the bacteriophage was the carrier of its genetic determination Why Must DNA Be Replicated? So that the information it holds can be maintained and passed to future cell generations To maintain the genetic information Three Modes of DNA Replication 1. Semiconservative Replication - Replicated DNA would consist of one “old” and one “new” strand  -Each DNA strand would serve as a template to create a complementary strand During DNA replication, the double helix would open and each original strand would serve as template for creation of new strand - End result: production of two DNA molecules - At least one strand is conserved 2. Conservative Replication - Two newly created strands are brought together and the parental strands reassociate - The original DNA molecule remains intact and an entirely new molecule is synthesized alongside it After the DNA replication, one DNA molecule is composed of original strands and the other is entirely composed of newly synthesized strand - Among the three, the DNA replication follows this model Each daughter DNA molecule contains one strand from the parent DNA molecule and one newly synthesized strand Ensures the preservation of genetic information and accurate transmission of genetic material to daughter cells especially during cell division 3. Dispersive Replication - Each strand would consist of both old and new DNA strand - Old and new DNA is dispersed in each strand Overview of DNA Replication Occurs during S phase Parent DNA molecule will unbind/unzip and separate the two strands apart from each other Step-by-Step Process of DNA Replication - Human DNA replicates at a rate of about 50 bases per second - In a DNA, there is a site where it is locally opened (replication fork) 1. Before replication, the helicase/unzipping enzyme binds to origin and separates strands Helicase untwists the double helix and break the hydrogen bonds holding the two strands together, resulting to two separate strands Each strand will be used as a template for the synthesis of the new strand 2. Binding proteins keep strands apart Helpful as they keep the strands apart so that they will not bind again 3. Primase makes a short stretch of RNA on the DNA template Replication begins with RNA primers which are short pieces of RNA ➔ Synthesized by the primase enzyme ➔ Synthesized when primase attracts the complementary RNA nucleotides 4. DNA polymerase adds DNA nucleotides to the RNA primer Uses the RNA primer as the starting point for adding nucleotides The DNA polymerase should add the correct complementary nucleotides of that of the template strands 5. DNA polymerase proofreading activity checks and replaces incorrect bases If ever the wrong base is added, it will check and replace it with the correct base 6. Continuous strand synthesis continues in a 5’ to 3’ direction Because of the DNA’s antiparallel configuration, DNA polymerase can only synthesize the DNA in the 5’ to 3’ direction Template: oriented in the 3’ to 5’ direction towards the replication fork DNA polymerase will also move in the template strand in 3’ to 5’ order towards the replication fork DNA polymerase will add the nucleotides in the 5’ to 3’ order in the new strand 7. Discontinuous synthesis produce Okazaki fragments on the 5’ to 3’ template The other template on the opposite strand, runs in the opposite direction because of the antiparallel configuration of the DNA molecule The other strand cannot be used by DNA polymerase to synthesize a continuous unbroken strand of the DNA in the 5’ to 3’ order Template: running from 5’ to 3’ order To make the new strand from the 5’ to 3’ direction, the synthesis should proceed backwards ➔ Replication happens away from the replication fork  Production of Okazaki fragments only happen in the lagging or discontinuous strand 8. Enzymes remove RNA primers. Ligase seals sugar-phosphate backbone The lagging strand will be completed when the DNA polymerase removes the RNA fibers from the Okazaki fragments As it removes the RNA primers from the Okazaki fragments, it will fill in the missing segment with the correct complementary DNA nucleotides Ligase: New enzyme will takeover ❖ Help seal the sugar-phosphate backbone DNA Amplification - Laboratory technique used to produce multiple copies of a specific DNA sequence - DNA replication conducted outside cells Polymerase Chain Reaction (PCR) - 1st best known DNA amplification technique - Uses DNA polymerase to rapidly replicate a specific DNA sequence in a test tube - Essentially, uses the same events that happens in DNA replication Requirements in the PCR Method 1. Target DNA sequence 2. Two types of laboratory-made primers - Single-stranded DNA molecule - Should be complementary in sequence to opposite ends of the target DNA 3. Make many copies of the 4 types of DNA nucleotides (adenine, thymine, cytosine, and guanine) 4. Special DNA polymerase - Comes from a thermophilic bacteria: bacteria that are heat-loving; can resist hot environment - Ex: Thermus aquaticus (Taq polymerase) and Thermococcus litoralis (Vent polymerase) Three Basic Steps of PCR Method - Standard lab method applied in molecular biology for the amplification of specific segments of double-stranded DNA - Based on the mechanisms of DNA replication, though it is performed in vitro - Underlying Principle:  ❖ The double-stranded DNA, which serves as the template in the reaction, is separated by heat ❖ DNA polymerase synthesizes a new daughter strand on each single strand in one direction ❖ By repeating these reactions continuously, a defined segment of DNA, the target, is amplified exponentially ➔ This and the high specificity of the reaction enable the amplification of target DNA segments 1. Denaturation - Heat will be the one to separate the two strands together - Heat required at 94 degrees celsius - Thermal cycler: automatically initiates the cycling temperature changes 2. Priming - Thermal cycler is set at 50 degrees celsius to 65 degrees celsius - Primer is used as a starting point for the making of new strand that is complementary 3. Extension - Heat required at 72 degrees celsius - With the help of the DNA polymerase, nucleotide is added which has bases that are complementary with that of the template strand Repeated many times to create many copies of DNA molecules Uses or Applications of PCR 1. Genetic Testing and Diagnostics - Identify genetic markers associated with disease, detection of mutation, identification of pathogens, and screening of genetic disorders 2. Forensics - Identification of suspects or victims and provide evidence for criminal investigations - Enables the amplification of minute amounts of DNA found in the crime scene 3. Disease Diagnosis - Diagnose infectious diseases caused by microorganisms (bacteria, viruses, or parasites) - Allows rapid and accurate detection of the genetic material of pathogen present in the sample of the patient 4. Biomedical Research - Enables the study of gene expression patterns, genetic variations, DNA sequencing, genotyping, etc - Cancer research, pharmacogenomics, developmental biology 5. Environmental Monitoring - Detection and identification of microorganisms in soil, water, and air samples 6. Paternity and Relationship Testing - Establish biological relationships 7. Ancient DNA Studies - Extraction and amplification of DNA from ancient samples - Instrumental in the study of genetic history particularly in the study of extinct species DNA Sequencing - Determines the order of the four chemical building blocks (bases) that make up the DNA molecule Basis for DNA Sequencing - Complementary base pairing Adenine (A) Thymine (T) Cytosine (C) Guanine (G) ❖ Basis for the mechanism by which the DNA molecules are copied when cell divides ❖ Underlies the method by which almost all DNA sequencing experiments are done ❖ Human genome has about 3 billion base pairs which spell out the instructions for making and maintaining a human being Methods Used for DNA Sequencing 1. Sanger sequencing - Invented by Frederick Sanger in 1977, which is a way to determine the base sequence of a small piece of DNA - Generates a series of DNA fragments (about 900 base pairs in length) of identical sequence that are complementary to the DNA sequence of interest - AKA chain termination method: dideoxynucleotides (ddNTPs) are added, terminating the DNA synthesis - Uses ddNTPs to determine the order/sequence of nucleotides in a nucleic acid Requirements: 1. Template to be sequenced - DNA template 2. Primers - DNA polymerase and 4 DNA nucleotides 3. DNA polymerase 4. 4 DNA nucleotides (dNTPs = dATP, dTTP, dCTP, dGTP_ 5. Different ddNTPs (ddATP, ddTTP, ddCTP, ddGTP) each labels with a different color of dye 6. Sequencing reaction (thermal cycler) Steps in Sanger Sequencing 1. DNA Amplification - To be sequenced has to be amplified 2. DNA Denaturation - Heat is used to separate the DNA to produce a complementary strand and a template strand for DNA sequencing 3. Dispersion of primed DNA - A primer is added to the sample/target sequence - The primer joins the 5’ of the DNA - The primed DNA is added equally among four reaction vessels 4. Addition of DNA Polymerase, dNTPs, and ddNTPs - These three are added to the reaction vessel - Four of them in each vessel 5. Attachment of DNA Polymerase - DNA polymerase will attach to the nucleotides (dNTPs) in the template strand where the primer is located until it reaches the ddNTP  6. Chain Termination - When DNA polymerase reaches the ddNTP, it will terminate the DNA sequencing - Once the ddNTP is base-paired, the sequence is terminated because ddNTP does not have a hydroxyl group at the 3’ carbon - The DNA fragments is the result of chain termination has different lengths and are formed across all four reaction vessels 7. Denaturing Polyacrylamide Gel Electrophoresis - Polyacrylamide Gel Electrophoresis: An equipment used to sequence the DNA - The sample is added to the wells of the gel and we will observe that DNA migrates from the negative pole to the positive pole This is because the DNA is made of the sugar-phosphate backbone which is negatively charged which becomes attracted to the positive charge - In the electrophoresis, the smaller/lighter lengths of DNA will be the ones to first migrate to the positive/opposite charge - Sequencing Ladder: Bands that are seen along the length of the gel plate forming a ladder-like structure 8. Sequence is read from the bottom to the top of the plate 9. Number of bases per sequence: 300-400 (depending on the reagents and gel used) - 5’ AGCGTCCCTAAGTCAACTG 3’ Result in the complementary sequence of the DNA sample 2. Next-generation sequencing - Collectively referred to the most recent set of DNA sequencing technologies - Ability to sequence millions of small pieces at once that can handle much larger DNA molecules much faster than the Sanger sequencing - Hast many variations and they use different technologies Features 1. Highly parallel - Many sequencing reaction happens at the same time 2. Micro scale - Reactions are tiny and it is done on a chip 3. Fast - Faster than Sanger sequencing - Human genome is able to be sequenced in just under a day - Before NGS: sequencing of the bases of human genome took about 10 years 4. Low-cost - Cheaper than that of the Sanger sequencing 5. Shorter length - Not just only for longer bases but also for those with shorter length Variation of NGS: 1. Nanopore Sequencing - NGS sequencing platform  - Each nucleotide can be identified by a disruption in current as it passes through the pores at about 1000 bases per second - Uses grapheme: 1-atom-thick sheet of carbon; strong, very thin, and conducts electricity - DNA strand will be threaded through the nanopores and electricity is produced - Each of the 4 DNA base types will disrupt the electrical field in slightly different way There is an algorithm which converts the voltage changes into the DNA sequence that they represent - The voltage approach is also used in semiconductor ships - 1st line = C; 2nd line = A; 3rd line = T; 4th line = G Uses/Applications of DNA Sequencing 1. Genome Sequencing - Provide comprehensive information about an organism’s genetic makeup - This has advanced the understanding of genetics, evolution, and the identification of disease-causing genetic variations 2. Medical Diagnostics and Personalized Medicine - Diagnosing genetic diseases and identifying disease-causing mutations 3. Cancer Genomics - Identification of genetic mutations associated with the development and progression of tumors 4. Forensics and Human Identification - Analysis of DNA from crime scene samples or unidentified human remains and establishing genetic relationships 5. Evolutionary Studies and Phylogenetics - Reconstructing evolutionary relationships between organisms and studying their genetic diversity 6. Metagenomics and Microbial Ecology - Analysis of complex microbial communities present in various environments 7. Pharmacogenomics - Identification of genetic variations affecting an individual’s response to medications - Helps optimize in the choosing of medications, dosage, treatment strategies for personalized medicines 8. Ancient DNA Studies - Providing insights into the genetic history and evolution of extinct species, including human ancestors