MolCellEng_Notes4

MolCellEng_Notes4 - 1 1. Chemical Synthesis of DNA 1....

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1. Chemical Synthesis of DNA 1. Oligonucleotide = DNA synthesized from scratch for use as PCR primers or probes for their sequence complements in a complex mixture of DNA; single-stranded segment that is synthesized in vitro (outside of organism) 2. Uses phosphoramidite monomers – carrying a DMT (dimethoxytrityl) group at 5’ and a cyanoethyl and a diisopropyl amino group at 3’ 3. Bases of the monomers are protected by benzoyl (A,C) or isobutyryl (G) group 4. STEPS 1. Detritylation – removes DMT group (5’ end) to get OH in place 2. Coupling – adds new monomer 3. Oxidation – to oxidate phosphate group 4. Repeat from STEP 1 and iterate until all monomers are coupled 5. Once last monomer is coupled, after oxidation, perform deprotection to remove cyanoethyl groups and to detach the synthesized DNA from solid support 6. Finish with detritylation 7. ****AFTER STEP 2, strands that are not coupled with new monomers will be capped (converting DMT to acetyl group) to prevent coupling in later cycles. 5. Oligonucletodies of up to 200 bp can be produced (# 21, Miterm Ex. 1) 6. DNA strands are produced from 3’ to 5’ end 7. Single stranded DNA molecules are produced 8. In deprotection step, cyanoethyl groups are removed and synthesized DNA is detached from solid support 2. DNA Sequencing 2
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1. Sanger Sequencing, aka chain termination method, aka sequencing by synthesis 1.
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MolCellEng_Notes4 - 1 1. Chemical Synthesis of DNA 1....

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