Chapter 4: Concept in Molecular Biology

Chapter 4 **Concept in Molecular Biology** DNA is the genetic material responsible for trait inheritance from generation to generation. DNA is composed of nucleic acid. Nucleic acid is composed of five carbon sugars, acidic phosphate and four types of nitrogen rich base, two forms of nucleic acids...

Chapter 4 **Concept in Molecular Biology** DNA is the genetic material responsible for trait inheritance from generation to generation. DNA is composed of nucleic acid. Nucleic acid is composed of five carbon sugars, acidic phosphate and four types of nitrogen rich base, two forms of nucleic acids were differentiated by their sugar composition; RNA contains ribose and DNA contains 2-deoxyribose, adenine, guanine, cytosine, uracil and thymine where uracil is found in RNA instead of thymine which is found in DNA. Adenine is always bound to thymine while guanine is always bound to cytosine. DNA carries the gene which carries the trait. The four bases (A, C, G and T) are the bases of codon which is composed of 21 amino acids. DNA is self-replicating and is transcribed into mRNA which is responsible for the synthesis of proteins. Reverse transcription is the procedure of synthesis of DNA from RNA. mRNAs can be transcribed to its complementary DNA (cDNA) and studied by recombinant DNA technique. The central dogma qualifications include genes that are not fixed, there are many transposable genetic elements in the eukaryotic genome, DNA sequence and protein amino acid sequence are not entirely collinear. Coding sequence exons are interrupted by non-coding sequence introns. Some RNA molecules display catalytic activity by RNA interference (small RNA molecules help regulate gene expression). **Recombinant DNA** Recombinant DNA is a part of DNA from donor organism pasted in the vector DNA to form new DNA. This technique is called molecular cloning. Cloning is a reproduction of daughter cell from one single cell by fission or mitotic division giving rise to genetically identical clone. **DNA Cloning** The essential tool for molecular cloning: restriction endonucleases, gel electrophoresis, vectors and host cells. Restriction endonucleases, enzymes cleave DNA at specific sequences and allow scientists to cut and paste DNA in a controlled and predetermined fashion, restriction endonucleases are isolated from bacteria, these enzymes are classified as type 1, 2, and 3 according to their mechanism of action. Gel electrophoresis is another technique for DNA cloning which allows the isolation and purification of DNA fragments of a defined length. It is used in molecular cloning to isolate and purify the vector and insert that which will be used to form a recombinant DNA vector. A vector is DNA molecule used to carry a foreign DNA fragment into a host organism which is used to produce large quantities of target DNA fragments or to get a foreign gene expressed in a host cell. Plasmids are bacterial circular genetic element that replicate independently from the chromosome. The Polymerase Chain Reaction (PCR) is an alternative method to cloning and isolating large amount of single DNA fragment or gene. 1. The central dogma of molecular biology: DNA → RNA→ protein. 2. Proteins have structural, enzymatic, and gene regulatory function. Through these mechanisms the genotype of a cell is translated into its phenotype. 3. DNA is the genetic material. 4. DNA is a double helix, consisting of two anti parallel strands of stacked nucleotides paired through hydrogen bonds. Adenine (A) always pairs with thymine (T), and cytosine (C) always pairs with guanine (G). 5. The structure of DNA determines its function. The sequence of nucleotides of one strand determines the sequence of its complementary strand, the basis for the semi conservative way of replication. 6. A gene is transcribed into precursor RNA, and the spliced mRNA is translated into the amino acid sequence of the coded protein. The sequence of mRNA unequivocally determines the sequence of the protein. 7. Recombinant DNA is the DNA of one organism 'cut and pasted' into a carrier vector. The foreign gene introduced in a host organism is functional because the genetic code is universal. 8. By DNA cloning, recombinant genes of complex animals, such as humans, are introduced into simple organisms, such as bacteria, and other model organisms, such as mice, allowing structural and functional studies. 9. Restriction endonucleases, bacterial enzymes that recognize and cut specific DNA sequences, are fundamental tools for DNA cloning. 10. Gel electrophoresis separates nucleic acids by size. 11. The most common host cell is the bacterium E. Coli. 12. Plasmids, the most commonly used vectors, are independently replicating circular DNA molecules modified to provide the host cell with resistance to antibiotics (selectable marker) and one or more restriction enzyme sites for inserting the recombinant gene. 13. By reverse transcription, mRNA is transcribed into complementary DNA (cDNA). 14. Automated fluorescent DNA sequencing based on the Sanger method is a standard laboratory procedure. 15. DNA sequencing of a cloned cDNA corresponding to a given gene is the easiest way to determine the amino acid sequence of a protein. 16. Genomic and cDNA libraries are collections of clones containing the genetic material of a cell. 17. Base pairing between complementary strands of DNA or RNA (hybridization with a labelled probe) is used to detect specific nucleic acid sequences in complex mixtures. 18. Southern blotting, Northern blotting, and dot blotting are hybridization-based techniques for nucleic acid sequence specific recognition. 19. The polymerase chain reaction (PCR) is an in-vitro method for DNA amplification. 20. Molecular polymorphism is studied at the genetic level by DNA typing. Methods for DNA typing relevant for transfusion medicine are restriction fragment length polymorphism, allele-specific oligonucleotide probe hybridization, allele-specific PCR amplification, DNA sequencing, and DNA profiling (DNA finger printing). 21. PCR is used for the early detection of transfusion-transmitted pathogens. 22. Other therapeutic uses of molecular biology are gene therapy and the clinical use of recombinant proteins, such as interferons, coagulation factors, and growth factors. 23. Molecular RBC antigen typing is used to either confirm serologic testing or in cases where serology is not possible or is not sensitive enough or where discrepancies occur. Review Questions: 1\. The central dogma of molecular biology states that: 2\. Recombinant-DNA technology is possible because: 3\. Agarose gel electrophoresis is a technique used for: 4\. Restriction fragment length polymorphism (RFLP) is based on the use of the enzymes: 5\. The polymerase chain reaction (PCR): 6\. Plasmids are: 7\. Some model organisms: 8\. DNA sequencing: 9\. RFLP and SSP are techniques used for: 10\. Recombinant DNA techniques: 11\. Transcription mediated amplification: 12\. Preseroconversion window: 13\. Red blood cell molecular antigen typing is useful in all listed situations except: Chapter 4 **Review Questions** **1.** a **2.** c **3.** c **4.** b **5.** d **6.** a **7.** d **8.** d **9.** c **10.** b **11.** b **12.** d **13.** c