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9 Cards in this Set
- Front
- Back
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Describe basic requirements for chain termination/Sanger sequencing
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1. Sequence specific DNA primer
2. DNA polymerase 3. 4 dideoxynucleotides for all 4 DNA nucleotides 4. High resolution method for fractionation of DNA sequences 5. Regular deoxynucleotides |
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How can DNA sequencing be applied to diagnosing and preventing human disease
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-To detect BRCA 1 gene mutations in predicting breast cancer incidence
-Sequencing pathogenic genomes. Ex. E-Coli bacteria in Germany -Personal genome sequencing |
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Basic requirements from PCR?
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1. Template DNA
2. Sequence specific DNA polymerase to flank target sequence 3. Deoxynucleotides (dNTPs) 4. Heat stable DNA polymerase that can survive repeated heating to 95 degrees - usually Taq polymerase Describe the procedure... |
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Uses of PCR for diagnosis and prevention of disease
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1. Rapid detection of pathogens ex. MRSA using real time PCR
2. To detect her2/neu amplification in breast cancer to see if herceptin can be used in tx 3. Detecting triplet repeat lengths for diseases such as myotonic dystrophy, huntington's and fragile X 4. Pre-screening of genotype to determine appropriate drug dosing ex. Warfarin dosage |
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Describe FISH procedure for chromosomal analysis
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1. DNA unmasking by digesting proteins and finding target DNA
2. Probe and target denaturation 3. Probe and target hybridization 4. Detection by bound reporter-flourochrome antibody 5. Image analysis on flourescent microscope Describe uses of FISH procedure... |
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Dideoxynucleotide (ddNTPs)structure/mechanism
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Compared to normal nucleotide which have 3' OHs, dideoxynucleotides have lack the 3' OH so that there is no mechanism to extend the DNA phosphodiester bond
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What are some improvements of the Sanger/DNA termination method
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1. Flourescently labelled ddNTPs
2. Capillary electrophoresis 3. Massively parallel sequencing (prod massive amts but very short sequencing reads which help if comparing new to old sequences) a. detection of light - pyro sequencing b. detection of protons - ion torrent (rxn occuring in 2 million dimples on a plate) |
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Describe the PCR procedure
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1. 95 degree heat denatures template
2. Excess molar primers anneal to both strands at 60 degrees 3. 72 degrees for Taq polymerase to do its thing ***Every cycle doubles the number of DNA product. So 30 cycles = 2^30 number of dna products |
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Describe the FISH procedure
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For determining karyotype and alterations in chromosome structure at the single cell level.
Uses: 1. Detecting trisomies in amniotic fluid 2. Detection of large scale deletions ex. DiGeorge 3. Gene amplification in breast tumors 4. Detection of translocations |
