Basic genomis Flashcards
Mention two methods developed in mid /1970’s that you can use to sequence DNA
- Chemical Method rarely used devoloped by Maxam - Gilbert.
- Sanger method or also known as a dideoxy or enzymatic developed by Frederick Sanger. This method is still used today with a little change to the basic method although great improvement have been made in efficiency and automation. Sanger Method is considerate a gold standard and the best method to estimate the new generation of DNA.
To carry out the Sanger method, it is necessary to implement the following requirements:
Requirement:
- Template DNA (to be sequenced) which is typically purified with plasmid and insert DNA.
- Specific primer DNA (~20 nucleotides long)
- DNA polymerase
- Deoxy-nucleotides =dNTPs (dATP, dGTP, dCTP, dTTP)
- Dideoxy nucleotides =ddNTPs (ddATP, ddGTP, ddCTP, ddTTP) low concentration called terminators.
Describe in steps the procedure carried out by Singer Method
Steps:
- DNA and primers heated to denature then cooled to anneal.
- Polymerase adds nucleotides specific to the template on end of primer.
- Occasionally a ddNTP is incorporated and the enzyme stops.
- Fragments of different lenghts are separated and then sequence is read.
What are the difference between the original and modern method?
Original method:
1. 4 reactions run, 1 with each ddNTP
2. Fragments separated on polyacrylamide slab gel , 1 line per ddNTP.
3. Entire gel dried and exposed to X-ray film.
4. Sequence interpreted from band order by human inspection.
Modern method:
1. All 4 fluorescently - labeled ddNTP used in 1 reaction each a different color.
What is the biggest problem in genomics?
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There are two kind of methods for sequencing larger pieces of DNA. Mention the main of each method and the pro and con.
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How big is a bacterial genomes, vertebrate genomes and in a scale of individual genes?
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Define C-value and the C-value paradox.
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Describe the two methods that could be used to sequence a genome. Mention the pro and con each one.
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Mention briefly the three methods to be used to identify the location of genes.
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What else we can find when we are sequencing genomes?
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What and why was the first complete geneome sequence of a free living organism? When was it published and genome size?
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What and why was the first complete geneome sequence of a free living organism using WSF before human genome? When was it published and genome size?
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When was it published the human genome? What is the genome size? Which are the two independent groups that worked on the human genome? How they worked?
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What is transcriptomics? Describe the EST sequencing, pro and con.
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What is the Microarrays method? How you can measure gene expression? Mention the two main approaches that are used to determine relative expression differences.
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What is the Affymetrix affy gene chips? Describe the procedure, Pro and con.
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What is the SAGE? Describe the procedure, Pro and con.
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What is the RNAq? Describe the procedure, Pro and con.
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Which fields can investigate the function of a particular gene?
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Define the main of Forward genetics and its procedure.
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Define the main of Reverse genetics. Describe the different kind of methods that could be implemented to carry out it.
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Define the main of Site directed mutagenesis and transgenics method and its procedure.
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What is interactomics, complementation groups, modifier screens?
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