Lec16+notes - Biochemistry & Genomics A gene sequence...

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1 A gene sequence reasonably predicts a protein sequence DNA is easier to sequence than protein or RNA How to sequence a genome – fast! What evolution tells us about protein structure and function Following expression of all genes at once allows complex regulatory circuits to be followed
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2 Next two lectures: Application of Biochemical Knowledge We have already learned: – DNAMAKESRNAMAKESPROTEIN – the code – how lots of enzymes that act on DNA work – if we could get the DNA sequence of all the DNA in an organism and find all the genes , we would know the complete set of proteins an organism needs to make.
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3 Current sequencing methods depend on >50 years of work leading to: • basic knowledge of enzymatic DNA replication and repair • ability to chemically synthesize short DNA sequences of defined sequence • ability to purify and amplify small pieces of DNA (cloning and PCR) • ability to create chemical analogs of nucleotides (“dideoxys”) • ability to separate DNA molecules that differ by one nucleotide in length (gel and capillary electrophoresis) • ability to attach dyes to nucleotides and detect the dyes with lasers • ability to attach DNA molecules to a solid surface • ability to capture images with very faint spots • computers and programs to manage large amounts of data
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4 DNA Synthesis reminder Template Primer DNA polymerase dNTP(s) basic knowledge of enzymatic DNA replication and repair
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5 ability to chemically synthesize short DNA sequences of defined sequence
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6 ability to purify and amplify small pieces of DNA (cloning)
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7 PCR, P olymerase C hain R eaction +DNA pol +dNTPs Major product Molecules #starting molecules x 2 #cycles (2 40 = 10 12 ) ability to purify and amplify small pieces of DNA (PCR)
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8 We can make the 4 things we need: DNA polymerase DNA template DNA primer DNA precursors, dATP, dGTP, dCTP and dTTP Now we need just a few more things before we can sequence.
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9 ability to create chemical analogs of nucleotides (“dideoxys”) ability to separate DNA molecules that differ by one nucleotide in length (gel and capillary electrophoresis)
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10 red A G T C green blue yellow ability to attach dyes to nucleotides and detect the dyes with lasers
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11 ability to attach DNA molecules to a solid surface
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12 Illumina uses dNTPs that chain terminate and have dyes, but these can be cleaved to allow subsequent bases to be determined from the same DNA.
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13 ability to attach DNA molecules to a solid surface
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14 Enzyme cascade for pyrosequencing (454)
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15 ability to capture images with very faint spots
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16 computers and programs to manage large amounts of data
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17 Single Molecule Real Time Sequencing (aka “SMRT”) Pacific Biosciences http://www.dnatube.com/video/3003/SMRT-DNA-Sequencing
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18 >RockyHorror Phage 12 length=56694 numreads=14327 AGGCGTCCGTGTgTGTGACCCGGGggTAGGgAGGTTTGTAGTGCCGTTCGCCAACACGACTGTTCGGAATCGTCGCCGCGCTCAGGTTCGTCAGCGTGACGGTGATGCTCCATGCGCGTTGCGGATCACGGCGGATTGCCAGGCAC
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This note was uploaded on 01/17/2011 for the course BIOC 100A taught by Professor Harrynoller during the Fall '10 term at University of California, Santa Cruz.

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Lec16+notes - Biochemistry & Genomics A gene sequence...

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