Introduction to Molecular Biology

Dr. M. Ravichandran Department of Medical Microbiology and Parasitology PPSP, USM Ext: 4592

History of Molecular Biology
1866 1944 1953 1961 1977 1985 1997 2001

Study of inheritance DNA is the genetic material Structure of DNA Genetic code DNA sequencing (enzymatic method) Polymerase chain reaction Dolly- Sheep cloning Human genome

Gregor Mendel Avery Watson and Crick Nirenberg and Khorana Sanger Kary B. Mullis Ian Wilmut Venter- Celera Genomics

TGA ( UAA. UAG. Start codon Stop codon Up stream.Purines: Adenine and Guanine G ≡C Glossary Pyrimidines: Cytosine and Thymine (Uracil in RNA) A = T/ A = U Gene is a segment of DNA which codes for a protein or RNA Codon is a triplet of 3 nucleotides that represents amino acids or terminal signals Start codon ATG (AUG in RNA) Stop codon TAA. which can be translatable into protein. UGA in RNA) ATG CAA AAG AAA TTG CTC CCC ATC CTT ATC TGA Met Gln Lys Lys Leu Leu Pro Ile Leu Ile --Start Stop Open reading frame (ORF):DNA sequence contains series of codons. down stream 3 . TAG.

Transcription: synthesis of RNA using DNA as template Translation: Synthesis of Protein using RNA as template Biotechnology: A sub discipline of biology in which biochemistry based process such as DNA technology are used for practical purposes cDNA: DNA molecule that results from reverse transcriptase activity on RNA AA: Amino acid. approximate weight of one AA = 110 Daltons bp: base pair 1AA is coded by 3 bases of DNA (codon) Kbp: Kilo base pair (1000 base pair) Vector: cloning vehicle eg: plasmid Plasmid Example: pNAME Signal sequence: It is an AA sequence required for protein secretion 4 .

Standard Genetic Code 5 .

Prokaryotes vs Eukaryots Prokaryotes Simple Genes grouped in Operon Polycistronic-Multiple No Post translational modifications gene Eukaryotes Complex Genes have Introns. Exons. 5’cap and 3’ PolyA Monocistronic-Single gene Post translational modifications Eg: glycosylation 6 .

Gene expression in Prokaryots Promoter Gene DNA replication (DNA polymerase) DNA Transcription (RNA polymerase) Translation (protein synthesis) mRNA Protein 7 .

splicing Mature mRNA m7G Translation AAAAAA Protein 8 .Gene expression in eukaryots Promoter Exon Intron Exon Intron Exon DNA Transcription (RNA polymerase) Primary mRNA transcript AAAAAA Removal of introns.

Electrophoresis of DNA How do you see DNA? By spectrophotometer: OD 260 nm 1OD = 50ug of dsDNA 33ug of ssDNA 40ug of RNA 9 .

Agarose gel electrophoresis 10 .

Samples that have been mixed with the sample buffer is then load into each well 11 .

Electrophoresis of DNA 12 .

RNA or DNA complementary to mRNA Normal gene expression mRNA Clone desired DNA fragment or transfer antisense DNA/RNA into the cell Antisense molecule RNA or DNA ||||||||||||||||||| mRNA Protein No Protein expression Application: •Inhibition of HIV viral replication inside the infected cells •Block the expression of Onchogenes Do you want to know •Inhibition of tomato ripening gene in tomatoes about13RNAi .Antisense molecules Antisense molecule .

Injecting gene specific dsRNA Transfecting a cell with a plasmid containing with the specific gene sequence and two opposed promoters 14 . 2. which can be used to silence a gene specifically Gene silencing using RNAi can be done by two methods 1.RNAi RNAi – interference RNA.

RNA Interference (RNAi) Double stranded RNA is introduced into a cell Double stranded RNA is chopped into pieces by an enzyme Guide RNA 15 RISC – RNA induced silencing complex .

RNA Interference (RNAi) The pieces “guide” a set of proteins to the normal mRNA The normal mRNA is clipped into pieces and destroyed 16 .

Mechanism of RNAi 17 .

RNAi in zebra fish embryo No RNA i With RNAi 18 .

19 .

Hundreds of biopesticides and other agricultural products are now used to improve our food supply and to reduce our dependence on conventional chemical pesticides. Biotechnology foods such as longer lasting tomatoes and better-tasting carrots and peppers are in the market. 20 . Environmental biotechnology products make it possible to more efficiently clean up hazardous waste without the use of caustic chemicals or incineration.Some Facts About Biotechnology Over 200 million people worldwide are being benefited from biotechnology drug products and vaccines. Biotechnology is responsible for hundreds of medical diagnostic tests that keep the blood supply safe from the AIDS virus and detect other conditions early enough to be successfully treated. as well as afforded significant advances in anthropology and wildlife management. a biotech process. has dramatically improved criminal investigation and forensic medicine. DNA fingerprinting.

OLD & S. RW.Suggested readings • Principles of Gene Manipulation.B primrose • Genetics: TA Brown • Molecular Biology: David Freifelder 21 .An introduction to Genetic Engineering.

Albert Einstein 22 .

M.usm. Ravichandran Department of Medical Microbiology and Parasitology 23 . USM Ext: 4592 mravic@kb.Questions and suggestions Dr.