Nucleic Acids PDF

NUCLEIC ACIDS Genetics - Deals with the study of heredity, how organisms pass on information in their genes to create new generations of the same species, of variations of the original. 3 Areas of Genetics 1. Transmission Genetics - Examines the relationship between the transmission of...

NUCLEIC ACIDS Genetics - Deals with the study of heredity, how organisms pass on information in their genes to create new generations of the same species, of variations of the original. 3 Areas of Genetics 1. Transmission Genetics - Examines the relationship between the transmission of genes from parent to offspring and the outcome of the offspring’s traits. (brown-eyed parents produce blue- eyed child) 2. Molecular Genetics - To understand how the genetic material works at the molecular level (how DNA features expresses genes) 3. Population Genetics - Helps us understand how the processes such as natural selection, how genetic variation is related to an organism’s environment (different forms of genes/ variant form of gene) A gene is a molecular unit of heredity on a living organism. It holds the information to build and maintain an organism's cells and pass genetic traits to the offspring. DNA is the main genetic material found in all living organisms. In molecular biology and genetics, the genome is the entirety of an organism's hereditary information. They are encoded in DNA molecules. The total DNA content in a single cell is known as the ‘genome’ of the organism, but some organisms have only RNA so that their genome is the total quantity of RNA content. The total content of the nuclear in a DNA cell is called as ‘nuclear genome’ and the total DNA content in mitochondria is called as ‘mitochondrial genome’. In addition, genome may also comprise of non-chromosomal genetic elements such as viruses, plasmids and transposable elements. Genome Gene Definition A group of all genes compromising of It is a unit of heredity which is a haploid set of chromosomes. composed of DNA occupying a fixed position on a chromosome. Content It is the total DNA content in the cell. It is a segment or portion of DNA molecule. Protein It is not a code of protein, as it It is a code for protein. contains full DNA. Composition It consists of all base pairs in a cell. It only consists of a few base pairs. Study It is referred to as genomics It is referred to as genetics. Number An organism has only one genome. An organism has thousands of millions of genes. Nucleic Acids - Are macromolecules that store genetic information and synthesize proteins. - Composed of long chain of nucleotides. Functions of Nucleic Acids 1. Stores genetic information 2. Transfer genetic information 3. Protect genetic information Nucleosides - Are glycosamines that can be thought of as nucleotides without a phosphate group. Nucleotides - The basic building blocks of nucleic acids. - RNA and DNA are polymers made of long chains of nucleotides - Consist of sugar molecule, either ribose or deoxyribose, attached to a phosphate group and a nitrogen-containing base. - The nitrogenous bases: Adenine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). - Serve a number of functions outside of genetic information storage, as messengers and energy moving molecules. - Have functions related to cell signaling, metabolism, and enzyme reactions. Nucleoside Nucleotide Nitrogenous Base Nitrogenous Base Sugar (5-carbon carbohydrate ribose) Sugar (5-carbon carbohydrate ribose) - Phosphate Base order is called the sequence. Nitrogenous Bases - Are organic molecules that contain nitrogen and act as bases in chemical reactions. - Building blocks of DNA and RNA Kinds of Nitrogenous Bases 1. Purines – is a heterocyclic aromatic organic compound, that consist of a pyrimidine ring fused to an imidazole ring. 2. Pyrimidines – is a heterocyclic aromatic organic compound similar to benzene and pyridine, that contains 2 nitrogen atoms at positions 1 and 3 of the six-member ring. Ring DNA RNA Pyrimidines Single T- thymine U – uracil C- cytosine C- cytosine Purines Two G- guanine G – guanine A - adenine A - adenine TYPES OF NUCLEIC ACID Nucleic acids – are bearers of genetic code. It can either be DNA or RNA. DEOXYRIBONUCLEIC ACID DNA as the genetic material. The genetic material is the deoxyribonucleic acid (DNA), the code that controls much of your body’s form and function. Genes that do not function properly can cause disease. All organisms and every person have two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than one percent of the same total) are slightly different between people. Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each person’s unique physical features. CRITERIA OF GENETIC MATERIAL A. Information The genetic material must contain the information necessary to construct an entire organism. It must provide the blueprint to determine the inherited traits of an organism. B. Transmission During reproduction, the genetic material must be passed from parents to offspring. C. Replication Because the genetic material is passed from parents to offspring, and from mother cell to daughter cells during cell division, it must be copied. D. Variation Within any species, a significant amount of phenotypic variability occurs. For example, Mendel studied several characteristics in pea plants that varied among different plants. DNA – genetic blueprint for the cell DNA - DNA is a macromolecule of carbon, nitrogen, oxygen, phosphorous and hydrogen atoms. It is assembled in units of nucleotides that are composed of phosphorylated ribose sugar and a nitrogen base. - There are four nitrogen bases that make up the majority of DNA found in all organisms. - Linear assembly of nucleotides makes up one strand of DNA, two-strands comprises the double-helix structure of DNA - The double helical structure of DNA results from the physiochemical demands of the linear array of nucleotides. Both the specific sequence of nucleotides in the strand as well as the surrounding chemical microenvironment can affect the nature of the DNA helix. Structure of DNA - DNA or deoxyribonucleic acid is a type of molecule known as a nucleic acid. - It consists of a 5-carbon deoxyribose sugar, a phosphate, and a nitrogenous base. - Double-stranded DNA consists of two spiral nucleic acid chains that are twisted into a double helix shape. - This twisting allows DNA to be more compact. In order to fit within the nucleus, DNA is packed into tightly coiled structures called chromatin. - Chromatin condenses to form chromosomes during cell division. Prior to DNA replication, the chromatin loosens giving cell replication machinery access to the DNA strands. - Genome size refers the amount of DNA contained in a haploid genome expressed either in terms of the number of base pairs, kilobases (1 kb = 1000 bp), or megabases (1 mb = 1 000 000 bp), or as the mass of DNA in picograms (1 pg = 10 – 12 g) Types of DNA 1. Genomic DNA/ Nuclear DNA - Comprises the genome of an organism - Controls expression of the various traits in an organism to study the various functions of the DNA. 2. Mitochondrial DNA - DNA located in the mitochondria - Derived from the circular bacteria - Lead to maternally inherited diseases. - mtDNA does not change from parent to offspring. RIBONUCLEIC ACID (RNA) RNA – It holds the information to build and maintain an organism's cells and pass genetic traits to the offspring. - Is a polymeric molecule essential in various biological roles in coding, decoding, regulation, and expression of genes. - It is principally involved in the synthesis of proteins, carrying the messenger instructions from DNA, which itself contains the genetic instructions required for the development and maintenance of life. - The central dogma of biology suggests that the primary role of RNA is to convert the information stored in DNA into proteins. - RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA. - It is synthesized as a single strand rather than a double helix. - Can also pair with complementary single strands of DNA or RNA and form a double helix. - RNA is copied or transcribed, from DNA. RNA Structure Types of RNA 1. Messenger RNA (mRNA) This carries information from the nucleus to the ribosomes which are sites for protein synthesis. The coding sequence on the mRNA determines the amino acid sequence in the protein. The mRNA is straight molecule extends from the 5’ to 3’ end. It is transcribed form a DNA template. On the mRNA nucleotides are arranged into codons consisting of 3 bases each. Each such codon specifies an amino acid. 2. Transfer RNA (tRNA) It is a small chain of about 80 nucleotides. It transfers specific amino acid molecules to a growing polypeptide chain. It has a clover leaf model with 5 arms each with a specific function. The tRNA also has an anticodon region that can base pair with the codon region on the mRNA. 3. Ribosomal RNA (rRNA) It is synthesized in the nucleolus. In the cytoplasm, rRNA and protein combine together to form a nucleoprotein called a ribosomes. The ribosomes and mRNA bind to carry out protein synthesis. It is very abundant in the cell and forms about 80% of the total RNA. The ribosomal RNAs form two sub-units namely; large and small subunit. Central Dogma of Biology The central dogma states that the pattern of information that occurs most frequently in our cells is: a. From existing DNA to make new DNA (DNA replication) b. From DNA to make new RNA (transcription) c. From RNA to make new proteins (translation).  The central dogma of life explains the flow of genetic information, from DNA to RNA, to make a functional product, a protein. The central dogma suggests that DNA contains the information needed to make all our proteins, and that RNA is a messenger that carries this information to the ribosomes. DNA Replication DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. Replication is an essential process because, whenever a cell divides, the two new daughter cells must contain the same genetic information, or DNA, as the parent cell. DNA replication is the production of identical DNA helices from a single double- stranded DNA molecule. Each molecule consists of a strand from the original molecule and a newly formed strand. Prior to replication, the DNA uncoils and strands separate. A replication fork is formed which serves as a template for replication. Primers bind to the DNA and DNA polymerases add new nucleotide sequences in the 5′ to 3′ direction. This addition is continuous in the leading strand and fragmented in the lagging strand. Once elongation of the DNA strands is complete, the strands are checked for errors, repairs are made, and telomere sequences are added to the ends of the DNA. Why Replicate DNA? DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis, biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus, must be replicated to ensure that each new cell receives the correct number of chromosomes. The process of DNA duplication is called DNA replication. Replication follows several steps that involve multiple proteins called replication enzymes and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of interphase during the cell cycle. The process of DNA replication is vital for cell growth, repair, and reproduction in organisms. Replication Process: Step 1: Replication Fork Formation Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single strands. DNA has four bases called adenine (A), thymine (T), cytosine (C) and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds with guanine. To unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork. This area will be the template for replication to begin. DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached the DNA backbone. The 5' end has a phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group attached. This directionality is important for replication as it only progresses in the 5' to 3' direction. However, the replication fork is bi- directional; one strand is oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging strand). The two sides are therefore replicated with two different processes to accommodate the directional difference. Step 2: Primer Binding The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase. Step 3: Elongation Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous. The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed. Step 4: Termination Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end, replication produces two DNA molecules, each with one strand from the parent molecule and one new strand. Enzymes in DNA Replication DNA replications occur with enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include: 1. DNA helicase - Unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA. 2. DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication. 3. DNA polymerases - Catalizes the addition of deoxynucleotides - Synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands. - No DNA polymerase can initiate a new chain - Can only add nucleotide onto a pre-existing 3’ OH group - To start a new chain, a primer is required [Primer – short stretch (11-12 nucleotides complementary in base pairing to the template DNA) of RNA molecule to which DNA polymerase can attach the first nucleotide] 4. Topoisomerase or DNA Gyrase - Unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled. 5. Exonucleases - Group of enzymes that remove nucleotide bases from the end of a DNA chain. 6. DNA ligase - Joins DNA fragments together by forming phosphodiester bonds between nucleotides. ** Leading Strand 1. The leading strand is a single DNA strand that, during DNA replication, is replicated in the 3' – 5' direction (same direction as the replication fork). DNA is added to the leading strand continuously, one complementary base at a time. 2. The first one is called the leading strand. This is the parent strand of DNA which runs in the 3' to 5' direction toward the fork, and it's able to be replicated continuously by DNA polymerase. 3. DNA synthesis occurs continuously because there is always a free 3’ OH at the replication fork to which a new nucleotide can be added, primed only once, at the origin ** Lagging Strand 1. A lagging strand is the name for one of the two DNA strands in a double helix that is undergoing replication. 2. A lagging strand is one of two strands of DNA found at the replication fork, or junction, in the double helix; the other strand is called the leading strand. A lagging strand requires a slight delay before undergoing replication, and it must undergo replication discontinuously in small fragments. 3. Lagging-strand replication is discontinuous, with short Okazaki fragments being formed and later linked together. 4. DNA synthesis occurs discontinuously. No 3’ OH at the replication fork to which a new nucleotide can attach. 5. Primer synthesize RNA primers multiple times to provide 3’ OH groups for DNA pol III Okazaki Fragments - are short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication. The Okazaki fragments are important for DNA synthesis because there is no 3' to 5' strand of DNA for the polymerase to use as a continuous template. RNA Transcription - (uses DNA as a template) RNA synthesis, or transcription, is the process of transcribing DNA nucleotide sequence information into RNA sequence information. RNA synthesis is catalyzed by a large enzyme called RNA polymerase. Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins). RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule. Transcription ends in a process called termination. Transcription is the first step of gene expression. During this process, the DNA sequence of a gene is copied into RNA. Before transcription can take place, the DNA double helix must unwind near the gene that is getting transcribed. The region of opened-up DNA is called a transcription bubble. RNA Polymerases - synthesizes RNA by following a strand of DNA. RNA polymerase is an enzyme that is responsible for copying a DNA sequence into an RNA sequence, during the process of transcription. Phases in Transcription 1. Initiation To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region called the promoter. Basically, the promoter tells the polymerase where to "sit down" on the DNA and begin transcribing. 2. Elongation Once RNA polymerase is in position at the promoter, the next step of transcription— elongation—can begin. Basically, elongation is the stage when the RNA strand gets longer, thanks to the addition of new nucleotides. During elongation, RNA polymerase "walks" along one strand of DNA, known as the template strand, in the 3' to 5' direction. For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the 3' end of the RNA strand. 3. Termination RNA polymerase will keep transcribing until it gets signals to stop. The process of ending transcription is called termination, and it happens once the polymerase transcribes a sequence of DNA known as a terminator. After termination, transcription is finished. An RNA transcript that is ready to be used in translation is called a messenger RNA (mRNA). In bacteria, RNA transcripts are ready to be translated right after transcription. Protein Synthesis Protein synthesis is the process in which cells make proteins. It occurs in two stages: transcription and translation. Transcription is the transfer of genetic instructions in DNA to mRNA in the nucleus. Protein synthesis occurs in cellular structures called ribosomes, found outside the nucleus. The process by which genetic information is transferred from the nucleus to the ribosomes is called transcription. During transcription, a strand of ribonucleic acid (RNA) is synthesized. The synthesis of proteins takes two steps: transcription and translation. Transcription takes the information encoded in DNA and encodes it into mRNA, which heads out of the cell's nucleus and into the cytoplasm. During translation, the mRNA works with a ribosome and tRNA to synthesize proteins. Protein Synthesis Steps 1. Transcription is the synthesis of RNA from a DNA template where the code in the DNA is converted into a complementary RNA code. 2. Translation is the synthesis of a protein from a mRNA template where the code in the mRNA is converted into an amino acid sequence in a protein. One specific amino acid can correspond to more than one codon. The genetic code is said to be degenerate. CODON VS ANTICODON CODON A codon is a three-letter adjacent non-overlapping nucleotide combination. This means that the three (3) nucleotide’s information code for one codon. Example: CAU, the nucleotides are those containing a base C, a base A, and a base U. When these nucleotides are joined in the order, CAU,(and not CUA, UCA, ACU or any other combination), this codon codes for one kind of amino acid. Thus the mRNA codon is ‘translated’ or ‘converted’ into amino acid. ANTICODON An anticodon is complementary to the codon. Thus if the codon is CAU, the anticodon is GUA. If the codon is UAG, the anticodon is AUC. If the codon is UCU, the anticodon is AGA. The codon is within an mRNA molecule, and the anticodon is within another RNA molecule in the cytoplasm known as tRNA. The tRNA has an anticodon because it needs to bind with the mRNA that lies at the ribosome. With that, the tRNA is held in contact with the mRNA. THE GENETIC CODE A genetic code is a listing of the codon with their corresponding amino acids. There are 64 codons in a genetic code. Sixty-one of these code for amino acids, 3 codons code for stop signals (meaning ‘stop translating, the protein is finish’). Of the 61 codons that code for amino acids, one, the AUG codon for the amino acid methionine, code also for ‘start’ codon (meaning ‘start translating here’) DNA transfer information to mRNA in the form of a code defined by a sequence of nucleotide bases. MUTATION - A mutation is a change that occurs in our DNA sequence, due to mistakes when the DNA is copied. - Mutations can also occur as the result of exposure to environmental factors such as smoking, sunlight and radiation. - Mutations can occur during DNA replication if errors are made and not corrected in time. - Mutations are changes in the genetic sequence, and they are a main cause of diversity among organisms. - In biological systems that are capable of reproduction, we must first focus on whether they are heritable; specifically, some mutations affect only the individual that carries them, while others affect all the carrier organism's offspring, and further descendants. - For mutations to affect an organism's descendants, they must: 1) Occur in cells that produce the next generation, and 2) Affect the hereditary material. Ultimately, the interplay between inherited mutations and environmental pressures generates diversity among species. "Mutation" typically refers to a change that affects the nucleic acids. In cellular organisms, these nucleic acids are the building blocks of DNA, and in viruses they are the building blocks of either DNA or RNA. One way to think of DNA and RNA is that they are substances that carry the long-term memory of the information required for an organism's reproduction. Two major categories of mutations 1. Germline mutations occur in gametes. These mutations are especially significant because they can be transmitted to offspring and every cell in the offspring will have the mutation. 2. Somatic mutations occur in other cells of the body. These mutations may have little effect on the organism because they are confined to just one cell and its daughter cells. Somatic mutations cannot be passed on to offspring. Mutations also differ in the way that the genetic material is changed. Mutations may change the structure of a chromosome or just change a single nucleotide. Types of Mutations 1. Missense mutation This type of mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene 2. Nonsense mutation A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all. 3. Insertion An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by the gene may not function properly. 4. Deletion A deletion changes the number of DNA bases by removing a piece of DNA. Small deletions may remove one or a few base pairs within a gene, while larger deletions can remove an entire gene or several neighboring genes. The deleted DNA may alter the function of the resulting protein(s). 5. Duplication A duplication consists of a piece of DNA that is abnormally copied one or more times. This type of mutation may alter the function of the resulting protein. 7. Repeat expansion Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. GENETIC TESTING - A medical test to identify changes in genes, chromosomes or proteins. 1. Molecular Testing - Look for changes in 1 or more genes. o Targeted single variant – specific variant in one gene o Single gene – test for any genetic changes in one gene (rule out / confirm specific dx) o Gene panel – panel tests look for variants in more than 1 gene, pinpoint a dx (ex. Genetic causes of epilepsy) o Whole genome sequencing – used when single gene or panel testing has not provided a dx, or suspected condition is unclear 2. Chromosomal Test - Analyze whole chromosomes, deletion, duplication, insertion. 3. Gene Expression Test - Determines which gene is active. 4. Biochemical Test - Study the amount or activity level or proteins or enzymes that are produced from genes. Uses of Genetic Testing 1. Newborn Screening 2. Diagnostic Testing 3. Carrier Testing 4. Prenatal Testing 5. Preimplantation testing – detect genetic changes in the embryos using assisted reproductive techniques (IVF) 6. Predictive & Pre-symptomatic Testing – detects gene mutation disorders that appear after birth 7. Forensic Testing Specimen Samples for Genetic Testing 1. Blood 2. Hair 3. Skin 4. Amniotic fluid 6. Nails 7. Other body tissues Methods for DNA Testing  Polymerase Chain Reaction (PCR) – new and faster method of DNA Isolation  DNA Sequencing – to determine the sequence of nucleotides present in a DNA  Karyotyping – chromosome analysis  Microarrays – to determine whether an individual’s DNA contain a duplication, deletion, or large stretches of identical DNA. DNA Extraction DNA extraction is the technique used to isolate DNA in a biological sample. The extraction of DNA is pivotal to biotechnology. It is the starting point for numerous applications, ranging from fundamental research to routine diagnostic and therapeutic decision-making. Extraction and purification are also essential to determining the unique characteristics of DNA, including its size, shape, and function. Main purpose of DNA extraction or DNA isolation: to provide a pure DNA. Importance of DNA Isolation 1. Study the genetic causes of disease and development of diagnostics and drugs 2. Essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment 3. Essential for determining paternity 3 Basic Steps in DNA Isolation 1. Cell Lysis The nucleus and the cell are broken open, thus releasing DNA. This process involves mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular proteins and free DNA. 2. Precipitation Separates the freed DNA from the cellular debris. It involves use of sodium (Na+) ions to neutralize any negative charge in DNA molecules, making them less water soluble and more stable. Alcohol (e.g isopropanol or ethanol) is then added, causes precipitation of DNA from the aqueous solution since it does not dissolve in alcohol. 3. Purification Purification removes all the remaining cellular debris and unwanted material. Once the DNA is completely purified, it is usually dissolved in water again for convenient storage and handling. DNA Extraction Methods 1. Physical DNA Extraction Methods a. Magnetic Bead DNA Extraction b. Paper DNA Extraction 2. Chemical DNA Extraction Methods a. Organic DNA Extraction Methods a.1 Phenol-Chloroform DNA Extraction Method b. Inorganic DNA Extraction Methods a.1 Proteinase K DNA Extraction Method a.2 Salting Out Method a.3 Silica Gel- Based DNA Extraction Method Polymerase Chain Reaction - New and faster method for DNA isolation - Used to rapidly make millions to billions of copies of specific DNA sample. - A typical PCR consists of: o Initial Denaturation: The reaction temperature is increased to 95 °C and the reaction is incubated for 2–5 min (up to 10 min depending on enzyme characteristics and template complexity) to ensure that all complex, double- stranded DNA (dsDNA) molecules are separated into single strands for amplification. o Cycling:  Denaturation: The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA).  Annealing: The temperature is lowered to approximately 5 °C below the melting temperature (Tm) of the primers (often 45–60 °C) to promote primer binding to the template.  Extension: The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended. o Repeat: Steps 1–3 are performed in a cyclical manner, resulting in exponential amplification of the amplicon