Comet Assay, Also Called Single Cell Gel Electrophoresis (SCGE), Is a Sensitive and Rapid

Lec -2-

Genome

Genome is the genetic material of an organism. It consists of DNA (or RNA in RNA viruses), including all of its genes. Each genome contains all of the information needed to build and maintain that organism.

Chromosome: It is a structure that occurs within cells and that contains genetic material. In Eukaryotes cells possess multiple linear chromosomes contained in the nucleus. It is a molecule of (DNA) and protein. DNA is helix double-stranded, the two strands are joined together by hydrogen bonds between the bases. The bases therefore form base pairs. The base pairs are specific. A only binds to T (and T with A), and C only binds to G (and G with C). A molecule of DNA is a very long, coiled structure that contains many subunits known as genes. They are polymers (long chain molecules) made from nucleotides. Nucleotides have three parts to them:

- A phosphate group, which is negatively charged B- A pentose sugar, which has 5 carbon atoms in it. In RNA the sugar is ribose. In DNA the sugar is deoxyribose. C- A nitrogenous base. There are five different bases (guanine, adenine, thymine, and cytosine, uracil )The base thymine is found in DNA only and the base uracil is found in RA only.

Nucleotide

Nucleotide

DNA replication

DNA replication is the process of producing two identical copies from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is composed of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication. In a cell, DNA replication begins at specific locations, or origins of replication, in the genome. Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bidirectionally from the origin. Most prominently, DNA polymerase synthesizes the new DNA by adding complementary nucleotides to the template strand.

- Mechanism of replication: -First, the double stranded DNA must separate, or unwind. To do this - DNA gyrase (class II topoisomerase) is responsible for uncoiling the DNA ahead of the replication fork. - Helicase is responsible for unwinding the DNA at the replication fork. - Primase gets this started by laying down a short RNA primer on the unwound DNA. The primer is made of RNA, but is complementary to the DNA sequence. - DNA polymerase then takes over and makes DNA that is complementary to the unwound DNA. Note, all biological DNA synthesis occurs from the 5' to the 3' end. - DNA synthesis occurs on both strands of the unwound DNA. The synthesis that proceeds in the direction of the replication fork is the leading strand. The synthesis that proceeds in the opposite direction to the replication fork is the lagging strand. The lagging strand contains fragments. DNA synthesis is bidirectional: 2 replication forks form and proceeds in opposite directions (like an expanding bubble). -Finally, RNA primers are replaced with DNA by a special DNA polymerase. The Okazaki fragments in the lagging strands are then stitched together by DNA ligase. - DNA polymerase has proof-reading activity, which means it corrects any mistakes (mutations) it makes.

Enzymes for manipulating DNA

1- Ligases catalyze formation of a phosphodiester bond between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the another to permit joining of 2 DNA molecules together. It is repair single-stranded breaksthat arise in double-stranded DNA molecules.

2- Topoisomerase: It is enzyme change the conformation of DNA by removing supercoils. This enzyme initiates unwinding of the double helix by cutting one of the strands. 3- Helicase: This enzyme assists the unwinding by breaking hydrogen bond between DNA strand.

4-Nucleases:This enzyme cut, shorten or degrade nucleic acid molecules.

a- Exonucleases are enzymes that cleave nucleotides from an end of a polynucleotide chain. These enzymes hydrolyze phosphodiester bonds from either the 3' or 5' terminus of polynucleotide molecules. Examples - Exonuclease III.

b-Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain that connects the RNA or DNA tracts.

5- DNA Polymerase: It is make copies of DNA molecules. A DNA polymerase is an enzyme that assists in DNA replication. It is builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. These enzymes also performs proof-reading and error correction.

RNA dependant DNA polymerase: Reverse transcript

Makes DNA from RNA.

6-Phosphatase catalyses the hydrolysis of 5’ phosphate groups from DNA or RNA or single nucleotides e.g. Alkaline phosphatase are most active at alkaline pH.

7- Kinase: Has the reverse effect of alkaline phosphatase, adding phosphate groups onto free 5’termini. Catalyze transfer of γ phosphate of ATP molecule to the 5 terminus of DNA fragment.By combining a phosphatase with a kinase the 5’ end of DNA can be labeled with a labeled phosphate group.

8- Restriction enzymes, also called restriction endonucleases, It was discovered in bacteria. Bacteria use them as a defense mechanism to cut up the DNA of viruses or other foreign DNA. These enzymes cut DNA molecules in specific places, recognize short, specific sequences of DNA bases, length varies (most common are “4-base” and “6-base”) know as a restriction endonuclease recognition site and make breaks in the sugarphosphate backbone of the DNA.

- Most recognized sequences are palindromic, the sequence on one strand matches that of the complementary strand read in the opposite direction

- Placement of cut varies; some leave “sticky ends”, others “blunt ends”

1