DNA STRUCTURE In 1962, scientists Francis Crick and James Watson were awarded the Nobel prize for their roles in discovering the structure of DNA, which is an acronym for deoxyribonucleic acid Anything that is alive, from bacteria to elephants, has DNA. DNA stores genetic material and passes it on to the next generation. A copy of a living entitys DNA is passed to developing offspring. Once the DNA is passed to the developing offspring, it is used to make that offsprings body parts.
DNA STRUCTURE DNA is a huge molecule called a macromolecule. However, DNA fits into small cells because it is packed in a process called supercoiling, in which DNA is wrapped around proteins called nucleosomes. Proteins called histones hold the coils together. Strands of DNA are divided into chromosomes, a full set of which is stored in the nucleus of each cell. These chromosomes, which basically instruct how the entire body is built, are called genes. A gene determines how a specific trait will be expressed.
Parts of a ChromosomeA chromatid is one of the twoidentical copies of DNA making up aduplicated chromosome, which arejoined at their centromere, for theprocess of cell division (mitosis ormeiosis). They are called sisterchromatids so long as they arejoined by the centromeres. Whenthey separate (during anaphase ofmitosis and anaphase 2 ofmeiosis), the strands are calleddaughter chromosomes.In other words, a chromatid is "one-half of a replicated chromosome". Itshould not be confused with theploidy of an organism, which is thenumber of homologous versions ofa chromosome.
Parts of a ChromosomeThe centromere is the part ofa chromosome that links sisterchromatidsA telomere is a region ofrepetitive nucleotidesequences at the end of achromosome, which protectsthe end of the chromosomefrom deterioration or fromfusion with neighboringchromosomes. Its name isderived from the Greeknouns telos end and merοspart. Telomere regionsdeter the degradation ofgenes near the ends ofchromosomes by allowingchromosome ends toshorten, which necessarilyoccurs during chromosomereplication.
DNA STRUCTURE Chemically, DNA is made of three components: nitrogen-rich bases, deoxyribose sugars, and phosphates. When combined, these components form a nucleotide. Nucleotides come together in pairs to form a single molecule of DNA.
DNA STRUCTURE There are four nitrogen-rich bases. These are adenine, guanine, thymine, and cytosine. Adenine and guanine have purine bases, which means they are a compound of two rings. Thymine and cytosine have pyrimidine bases, which means they have a single six- sided ring structure. These rings stack up in DNA to make the molecule compact and strong. In order to make a complete nucleotide, the bases are attached to deoxyribose and a phosphate molecule. Nucleotides are the building blocks of DNA. To make a complete DNA molecule, these nucleotides join together to make matched pairs and form long double strands called double helixes.
Nitrogenous bases are typically classifiedas the derivatives of two parentcompounds, pyrimidine and purine. Theyare non-polar and due to theiraromaticity, planar. Both pyrimidines andpurines resemble pyridine and are thusweak bases and relatively unreactivetowards electrophilic aromaticsubstitution. Their flat shape is particularlyimportant when considering their roles innucleic acids as nucleobases (buildingblocks of DNA and RNA):adenine, guanine, thymine, cytosine, anduracil. These nitrogenous bases hydrogenbond between opposing DNA strands toform the rungs of the "twisted ladder" ordouble helix of DNA or a biological catalystthat is found in the nucleotides. Adenine isalways paired with thymine, and guanine isalways paired with cytosine. Uracil is onlypresent in RNA: replacing thymine andpairing with adenine.
A nitrogen-containing ringstructure called a base. Thebase is attached to the 1carbon atom of the pentose.In DNA, four different basesare found:two purines, called adenine(A) and guanine (G)two pyrimidines, calledthymine (T) and cytosine (C)*A always pairs with T*C always pairs with G
DNA STRUCTURE A deoxyribonucleotide is the monomer, or single unit, of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts: a nitrogenous base, a deoxyribose sugar, and one phosphate group. The nitrogenous base is always bonded to the 1 carbon of the deoxyribose, which is distinguished from ribose by the presence of a proton on the 2 carbon rather than an -OH group. The phosphate groups bind to the 5 carbon of the sugar. When deoxyribonucleotides polymerize to form DNA, the phosphate group from one nucleotide will bond to the 3 carbon on another nucleotide, forming a phosphodiester bond via dehydration synthesis. New nucleotides are always added to the 3 carbon of the last nucleotide, so synthesis always proceeds from 5 to 3.
DNA replication is a biologicalprocess that occurs in all livingorganisms and copies their DNA; it isthe basis for biological inheritance.The process starts when one double-stranded DNA molecule producestwo identical copies of the molecule.
Each strand of the original double-strandedDNA molecule serves as template for theproduction of the complementary strand, aprocess referred to as semiconservativereplication.Cellularproofreading and error toe-checkingmechanisms ensure near perfect fidelity forDNA replication.DNA replication can also be performed invitro (artificially, outside a cell).
DNA polymerases, isolated fromcells, and artificial DNA primers are usedto initiate DNA synthesis at knownsequences in a template molecule.Thepolymerase chain reaction (PCR), acommon laboratory technique, employssuch artificial synthesis in a cyclicmanner to amplify a specific target DNAfragment from a pool of DNA.
Legend:The major types ofproteins, which must worktogether during the replication ofDNA, are illustrated,showing their positions.
When DNA replicates, many differentproteins work together to accomplish thefollowing steps:1. The two parent strands are unwound withthe help of DNA helicases.2. Single stranded DNA bindingproteins attach to the unwoundstrands, preventing them from winding backtogether.
3. The strands are held in position, bindingeasily to DNA polymerase, which catalyzes theelongation of the leading and lagging strands.(DNA polymerase also checks the accuracy ofits own work!).
4. While the DNA polymerase on theleading strand can operate in acontinuous fashion, RNA primer isneeded repeatedly on the lagging strandto facilitate synthesis of Okazakifragments.DNA primase, which is one ofseveral polypeptides bound together in agroup called primosomes, helps to buildthe primer.
5. Finally, each new Okazakifragment is attached to thecompleted portion of the laggingstrand in a reaction catalyzedby DNA ligase.
Amplification of DNA by fragment PCR• Introduction• The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours.
• While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods.
Basic PCR• The PCR process was originally developed to amplify short segments of a longer DNA molecule (Saiki et al. 1985). A typical amplification reaction includes target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
• Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time.• This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor of more than a million in a matter of hours.
• Each cycle of PCR includes steps for template denaturation, primer annealing and primer extension. The initial step denatures the target DNA by heating it to 94°C or higher for 15 seconds to 2 minutes. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase. In the next step of a cycle, the temperature is reduced to approximately 40– 60°C.
• At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C. The extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C for denaturation.
• Each step of the cycle should be optimized for each template and primer pair combination. If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. After 20–40 cycles, the amplified product may be analyzed for size, quantity, sequence, etc., or used in further experimental procedures.