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Computational Biology 2 Pawan Dhar BII Lecture 1 Introduction to terms, techniques and concepts in molecular biology Molecular biology - a primer Human body has 100 trillion cells each containing 3 billion pairs of nucleotides. xxxxxxxxxxxxx A 6 foot human has so much of DNA that it can travel to and fro, from Earth to Sun 600 times !!! RNA polymerase (yellow) attaches to a DNA, walks down the DNA and creates a copy of DNA into a strand of messenger RNA (blue) The mRNA strand floats free and finds a ribosome (green). Ribosome churns out a chain of amino acids (purple) Amino-acid chain folds into the protein Glossary Genome The entire genetic component of an organism ORF A series of codons starting with an initiation codon and ending with a termination (open reading frame) codon DNA sequencing The technique for determining the order of nucleotides in a DNA molecule DNA replication Synthesis of a new copy of DNA molecule DNA polymerase An enzyme that synthesizes DNA Cloning Inserting DNA fragment into cloning vector and propagating recombinant vector in a host organism Question time An ORF represents an open reading frame. Is there a CRF too ? (CRF = closed reading frame) Vectors Vector Insert size Lambda vector Cosmid, fosmid PI BAC, PAC YAC Mega-YAC 18 kb 40 kb 125 kb 300 kb 600 kb 1400 kb p=95% (human genome) 532 500 240 000 77 000 32 000 16 000 6 850 The size of the library depends on the cloning vector Restriction Enzymes DNA cutting enzymes found in bacteria. Example: Hae III 5’ G G C C 3’ 3’ C C G G 5’ Blunt ends EcoRI Sticky ends 5’ G A A T T C 3’ 3’ C T T A A G 5’ DNA ligase joins the broken ends n i RI n o io c E ct a Restriction map of pBR322 Question time A student repeatedly failed to cut a plasmid with two different restriction endonucleases, even though he was using the enzymes in good condition and the plasmid had sites for both. Plasmids 1. Extrachromosomal circular DNA molecules which are not part of the bacterial chromosome. 2. Size range: 1 - 200 kb 3. Carry functions advantageous to hosts e.g., (a) produce enzymes which degrade antibiotics or heavy metals (b) produce restriction and modifying enzymes. 4. Replication is coupled to host replication in both stringent (1-2 copies / division) and relaxed manners (10-200 copies / division). How do you insert foreign DNA into plasmid ? • Cut plasmid on a unique restriction site • Also cut the insert by the same restriction enzyme (preferably) • Incubate linearized plasmid with the fragment to be inserted • Check insertion by gel electrophoresis Finding a way into plasmid’s territory Question time Devise a strategy to ligate an “insert” with the “linearized plasmid” given that the insert has a blunt end and plasmid has a sticky end ? Transformation Insertion of recombinant plasmid into bacteria 1. Chemical: e.g., calcium phosphate mediated 2. Physical : e.g., electroporation, DNA gun 3. Viral: e.g., retroviruses Bacteria a living photocopy machine Producing insulin through genetic engineerin g Effect of plasmid size on transformation efficiency Molecule size (kb) 2.0 3.2 4.3 12.5 20.0 39.0 54.0 % Maximum probability 57 100 86 43 36 14 6 Question time Given the task of sequencing a large stretch of DNA, what plasmid size would you choose for transformation into bacteria and why ? Artificial vehicle acting naturally What is cloning ? A procedure to generate large number of copies of a single sequence of DNA Properties 1. The host will accept foreign DNA and can still complete its life cycle 2. Permit the host to divide normally and be distinguished from non transformed members of community cDNA cloning It is a method of obtaining a DNA copy of the mRNA and making a library out of it. STEPS 1. Bind oligo DT to the poly A tail of the mRNA 2. Add reverse transcriptase and make its cDNA copy 3. Digest mRNA and add poly C tail to 3’end of DNA 4. Add oligo dG and synthesize a complementary strand 5. Add dC’s to the 3’end of double stranded cDNA 6. Insert dC tailed DNA to dG tailed linearized plasmid 7. Transform E.coli cells 8. Use selection makers to identify transformed cells Question time Is there a way to prevent self-ligation of linearized plasmid ? If No explain why, if Yes explain how. Creatin g genomi cs What is a genomic library ? A genomic library contains DNA fragments which when aligned together represent the entire genome. The major problem Not all the fragments find their way into plasmids !!! Question time What strategy would you use to include the entire genome into plasmid library Chromosome walking It is a technique for constructing a clone contig by identifying overlapping fragments of cloned DNA Contig: is a continuous set of overlapping DNA sequences Question time What is the best way to “walk through” a highly repetitive DNA? PCR: Polymerase Chain Reaction Also known as “Molecular photocopying” Invented by Kary Mullis - mid 1980s The real story behind PCR …. Taq polymerase: Thermus aquaticus 3 steps: Denature, Anneal & Elongate PCR protocol - an overview Theory and Application of PCR PCR Animation follows * 92 - 94oC for 30 - 60 sec (denature) * 37 - 72oC for 30 - 60 sec (anneal) * 72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length) * 25 - 35 cycles only (otherwise enzyme decay causes artifacts) * 72oC for 5 min at end (to allow complete elongation of all product DNA) End of Human Genome lecture # Suggested Reading: Books 1. Genomes by TA Brown 2. Molecular Cell Biology by Lodish et al 3. Molecular and Cell Biology - Schaum series Research Papers 4. Trends Biotechnol 1994: 12, 280-86 5. Trends Biochem 2001: 26, 268-270