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 entity's DNA is passed to developing offspring. Once the DNA is passed to the developing offspring, it is used to make that offspring's body parts.

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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.

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Parts of a ChromosomeA chromatid is one of the two

identical copies of DNA making up a duplicated chromosome, which are joined at their centromere, for the process of cell division (mitosis or meiosis). They are called sister chromatids so long as they are joined by the centromeres. When they separate (during anaphase of mitosis and anaphase 2 of meiosis), the strands are called daughter chromosomes.In other words, a chromatid is "one-half of a replicated chromosome". It should not be confused with the ploidy of an organism, which is the number of homologous versions of a chromosome.

Parts of a ChromosomeThe centromere is the part of

a chromosome that links sister chromatids

A telomere is a region of repetitive nucleotide sequences at the end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. Its name is derived from the Greek nouns telos 'end' and merοs 'part.' Telomere regions deter the degradation of genes near the ends of chromosomes by allowing chromosome ends to shorten, which necessarily occurs during chromosome replication.

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.

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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.

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Nitrogenous bases are typically classified as the derivatives of two parent compounds, pyrimidine and purine. They are non-polar and due to their aromaticity, planar. Both pyrimidines and purines resemble pyridine and are thus weak bases and relatively unreactive towards electrophilic aromatic substitution. Their flat shape is particularly important when considering their roles in nucleic acids as nucleobases (building blocks of DNA and RNA): adenine, guanine, thymine, cytosine, and uracil. These nitrogenous bases hydrogen bond between opposing DNA strands to form the rungs of the "twisted ladder" or double helix of DNA or a biological catalyst that is found in the nucleotides. Adenine is always paired with thymine, and guanine is always paired with cytosine. Uracil is only present in RNA: replacing thymine and pairing with adenine.

A nitrogen-containing ring structure called a base. The base is attached to the 1' carbon atom of the pentose. In DNA, four different bases are found:

two purines, called adenine (A) and guanine (G)

two pyrimidines, called thymine (T) and cytosine (C)

*A always pairs with T

*C always pairs with G

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'.

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DNA replication

DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance.

The process starts when one double-stranded DNA molecule produces two identical copies of the molecule.

Each strand of the original double-stranded DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication.

Cellularproofreading and error toe-checking mechanisms ensure near perfect fidelity for DNA replication.

DNA replication can also be performed in vitro (artificially, outside a cell).

DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule.

Thepolymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA.

Different Proteins Needed in DNA Replication

Legend: The major types of proteins, which must work together during the replication of DNA, are illustrated, showing their positions.

When DNA replicates, many different proteins work together to accomplish the following steps:

1. The two parent strands are unwound with the help of DNA helicases.

2. Single stranded DNA binding proteins attach to the unwound strands, preventing them from winding back together.

3. The strands are held in position, binding easily to DNA polymerase, which catalyzes the elongation of the leading and lagging strands. (DNA polymerase also checks the accuracy of its own work!).

4. While the DNA polymerase on the leading strand can operate in a continuous fashion, RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments.DNA primase, which is one of several polypeptides bound together in a group called primosomes, helps to build the primer.

5. Finally, each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by 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.

PRESENTED BY: Marri UntiveroAstrid Dominguez