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Definition
| Prokaryotes: 70S ( small (30S) and a large (50S)) Eukaryotes: 80S (small (40S) and large (60S)) |
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Term
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Definition
- Svedberg units
- a measure of the rate of sedimentation of a particle in a centrifuge
- increase in sedimentation rate is associated with increase in size of the particle
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Term
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Definition
| protein + 16S rRNA (responsible for initiation and termination of transcription) |
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Term
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Definition
- Contains semi-universal regions that are identical between many species of bacteria
- Contains hypervariable regions that differ between species but are highly conserved within a species
- 16SrRNA gene sequencing has become a rapid, accurate alternative to phenotypic methods
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Term
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Definition
| The polymerase chain reaction was developed in order to mimic nature's own way of replicating DNA |
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Term
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Definition
- Unwinding – (94–98 °C) Denatures the dsDNA
- Annealing – (50–65 °C) Primers attach to complementary sequences
- Polymerization (75–80 °C ) - DNA polymerase lays down nucleotides
- Each cycle doubles number of molecules
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Term
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Definition
- DNA Polymerase
- Magnesium Chloride
- RNA Primers
- Template Strand
- dNTPs
- Buffer
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Term
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Definition
- Taq: DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus
- Lives in hot springs and hydrothermal vents
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Term
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Definition
- Thermostable polymerase requires magnesium as a cofactor.
- The success of a PCR is dependent on amount of magnesium.
- Catalyst: the magnesium is not actually consumed in the reaction but the reaction cannot proceed without magnesium.
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Term
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Definition
- Single-stranded DNA required for DNA synthesis. (in vivo they are RNA)
- Complementary to the 3' ends of each strand of the DNA target.
- Length < 30 nucleotides
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Term
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Definition
- Deoxyribonucleoside triphosphate
- Building blocks that DNA polymerase uses to synthesize a new DNA strand.
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Term
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Definition
- Electrophoresis – the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
- The electric field forces the fragments to migrate toward the positive potential due to the net negative charge of the phosphate backbone of DNA.
- The molecules will move through the matrix at different rates based on mass
- Small fragments move quickly, large fragments move slowly
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Term
| Components of Gel Electrophoresis |
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Definition
- Gel Matrix – TAE/TBE + agarose + Ethidium Bromide (EtBr). Porous to allow electric current and dna to pass through, solid enough to impede the movement of the fragment and keep it imbedded in the media when electricity has ceased.
- TAE = Tris base + acetic acid + EDTA Buffer – TAE/TBE (to match gel).
- Buffer has ions in it to allow electricity to pass through it.
- Sample – movement depends on size of product
- Loading Dye – Added to the sample to weigh it down and make it visible during loading
- Ladder – made of DNA fragments of known size. Used as a reference.
- Ethidium Bromide: Intercalating agent - When exposed to ultraviolet light, it will fluoresce with an orange color
- Known carcinogen – causes replication difficulties in DNA
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Term
| Sanger Method of DNA Sequencing |
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Definition
- Used on short segments of DNA – 850 bp or less
- DNA can be separated by size.
- dideoxyribonucleotide triphosphate (ddNTP): share the same structure as a normal dNTP except the 3' OH group, replaced by an H
- DNA Polymerase would be able to integrate this modified nucleotide if added in vitro
- Lack of a 3' OH terminates DNA synthesis
- Polymerase will randomly integrate either a dNTP or a ddNTP into the synthesizing strand if the ddNTP complements with the nucleotide on the template strand.
- In a solution where the same chain of DNA is being synthesized over and over again, the new chain will terminate at all positions where the nucleotide has the potential to be added because of the integration of the dideoxynucleotides
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Term
| High Throughput DNA Sequencing |
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Definition
- Amplify gene of interest with PCR – label dNTPs with unique fluorescent markers to determine the identity of the nucleotides within the sequence
- Also act as analogs that terminate replication once added
- Replaces gel electrophoresis with capillary electrophoresis.
- Fragments migrate toward the + anode end of the capillary, and a laser hits the fragment as it passes through
- The laser allows the four nucleotides to radiate different colors, causes the dye on the nucleotides to illuminate a different color.The camera then records these colors and sends the results to the computer for analysis.
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