Sanger DNA Sequencing Exam Questions and Answers Already Passed

Sanger DNA Sequencing Exam Questions and Answers Already Passed DNA Sequencing - Answers the primary method for gne and protein characterization Sanger Sequencing AKA - Answers - Dideoxynucleotide sequencing - Chain termination sequencing (DNA synthesis will terminate with the incorporation of a dideoxynucleotide b/c no more 5'-3' synthesis possible) Principle of Sanger Sequencing - Answers - All four dNTPs are used - Four separate reactions are set up - Each as a different ddNTP: ddATP, ddTTP, ddCTP, ddGTP - Klenow will incorporate dNTPs until a ddNTP is incorporated which terminates synthesis - DNA is denatured and the four smaples are loaded next to each other and DNA separted on a PAGE gel - Separate based on size of fragment -> run on a high % page gel to separate very specifically (even single base differences) Steps of sanger sequencing - Answers 1. PCR clean up (remove excess primer by exonucleas 1 and dNTP's by alkaine prophasphatase 2. Cycle sequencing 3. Purify the extension products: ethanol precipitation ' 4. sequencing analaysis ' 5. gel or capillaries 6. Analysis A-T cloning - Answers -In a PCR-reaction most polymerases add A to the end of the product -Vector has a T-overhang where the PCR-poduct is ligated. -Following ligation, the vector with the PCR-product is transformed into competent E.coli cells by heat-shock -The E.coli is plated on to selective agar plates -Pick colonies (clones) and culture -Isolate plasmid-DNA -Sequence to identify clones with insert Sequencing AMM - Answers -Identification of bacterial strains -Epidemiological studies -Genotyping of virus -Internal quality controls for in-house PCR -Verification of PCR positive results when implementing a new analysis -Unexpected results in PCR Sanger is useful - Answers -Useful tool with a lot of applications in a medical microbiology diagnostics lab -Suitable for shorter sequences -Bioinformatics is managable De Novo sequencing - Answers to identify new agent, De Novo sequencing is essential for the development of new diagnostic tools Sequences available in Genbank to develop new PCR's Sequencing of new influenza strains in connection with pandemics. - Answers De Novo sequencing is essential for the development of new diagnostic tools Sequences available in Genbank to develop new PCR's Sequencing of new influenza strains in connection with pandemics. - Answers NGS platforms - common features: - Answers Random fragmentation of starting DNA, ligation