chapter 19 lecture 13/14 Flashcards
probe
DNA or RNA with a base sequence complementary to a sequence in the gene of interest, and with a radioactive or chemiluminescent molecule attached allows visualization
Southern blot
DNA
northern blot
RNA
western blot
protein
Polymerase chain reaction
amplifying DNA fragments
gene cloning
amplifying a specific piece of DNA via a bacteria cell
cloning vector
a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell
PRC reaction
Taq polymerase: stable DNA polymerase at high temperature
- reverse transcription PCR
Reverse transcriptase PCR
important to get a copy of the coding sequence of the DNA
qPCR
incorporate a florescent probe that allows you to quantify how much code is present
Real-time PCR
quantitatively determining the amount of DNA amplified as the reaction proceeds
Multiplexing Real-time PCR
probes with different spectral wavelengths can be used to allow the quantitation of multiple targets in a single reaction
Gel electrophoresis
- dNA migrate to + electrode
- separation based on fragment length
- DNA cut with restriction enzyme
- gel soaked in ethidium bromide which sticks to DS DNA and fluoresces when exposed to UV light
restriction enzymes
recognizing and cutting DNA at specific nucleotide sequence
what do prokaryotes use to ward off viral parasites?
restrictions enzymes
Where do restriction enzymes come from?
exist naturally in bacteria, which use to prevent the entry of viral DNA
DNA fragments that are 500bp , 1000bp amd 2000 bp in length are separated by a gel electrophoresis. Which fragment will migrate farthest in the gel? a, 2000 bp fragments b. 1000 bp fragment c. 500 bp fragment d. all wllmigrate equal distance
c. 500 bp fragment
how do Northern and Western blotting differ fromsouthern blotting?
southern - used to transfer DNA from a gel to a solid medium
- Northern used to transfer RNA from a gel to a solid medium
- Western used to transfer protein from a gel to a solid medium
probe in southern and northern is a complementary sequence western is an antibody
cloning vector has what 3 characteristics
- an origin of replication : ensures vector replicated within the cell
- selectable markers: enable cells containing vector to be ID’d
- 1 or more unique restriction site into which a DNA fragment can be inserted
how is a gene inserted into a plasmid cloning vector?
gene and plasmid cut with same restriction enzyme and mixed together. DNA ligase is used to seal the nicks in the sugar backbone
Why is the use of a heat- stable DNA polymerase important to the success of PCR?
heat-stable DNA polymerase enzyme is important to the success of PCR because the first step of the reaction requires that the solution be heated to between 90 and 100 degrees C to separate the two DNA strands. Most enzymes are denatured at this temperature. with the use of a heat-stable polymerase, the enzyme can be added at the beginning of the reaction and will function throughout multiple cycles
briefly explain how synthetic probes are created to screen a DNA library when the protein encoded by the gene is known
with the use of the genetic code and the amino acid sequence of the protein, possible nucleotide sequences that might encode the protein, taking into consideration synonymous codons is used to probe the library. to minimize the number of sequences required, a region of the protein that has a relatively little degeneracy in its codons is selected
how are candidate genes identified by positional cloning evaluated to determine whether they encode the phenotype of interest
the expression pattern of the gene can be examined and the coding region of copies of the gene from individuals with the mutant phenotype can be compared with the coding region of wild-type individuals
