Chapter 8 Notes C
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What does TAQ polymerase usually do?
usually puts an A at the 3' end as an overhang, which will allow binding to a extrication enzyme with an exposed T overhang
What are the steps of the PCR?
Denaturation: heat strands to separate at 90 degrees Celsius
Annealing: hybridization of primers at 50-60 degrees Celsius
Elongation: DNA polymerase adds dNTPs; 72 degrees Celsius, which is optimal for TAQ polymerase
What is important about primers for cloning?
There is an overhang with restriction sites with becomes ds after the first cycle
used for directional cloning
what does the overhang primer allow?
It allows a restriction enzyme to be it reduced into the DNA sequence that will eventually be used for cleavage and later insertion I to a cloning vector
How to use PCR to obtain a genomic clone.
- To obtain a genomic clone:
- 1) chromosomal DNA is purified fromc ells
- 2) PCR primers that flank the stretch of DNA to be clooned are added, and many cycles of reaction are completed
- Since only the DNA between the primers is amplified, PCR is a way to obtain a short stretch of chromosomal DNA seelctively connects virtually pure form
How to use PCR to obtain a cDNA clone.
- mRNA is purified
- The first primer is added to teh population of mRNAs and reverse transcriiptase is used to make a complementary DNA starand. The second primer is then added, and the single-stranded cDNA molecule is amplified through many cycles of PCR.
Variable nucleotide repeats
regions in DNA that contain the same, short, repeated sequence sbut have different numbers
hypervariable microsatellite sequence
paternal and maternal variants are inherited, giving the offspring unique VNTRs
Real Time PCR
While PCR is running, after every sample, we spin it around so that the spec reads the SYBR green level, keepng track of it
What are the characteristics of the machine/ tubes?
- There is a thin walled glass capillar tube that is so thin and glass
- PCR is done more rapidly than normal
What are the differences between standard PCR and real time PCR?
1) Real time is quicker: warming and cooling by heating the block in old PCR
2) Change temp of air in chamber, making it more rapid
3) Amount of sample that can be put in can be 3-4 uL; helps with rapid temperature change
4) in PCR reaction mixture, in a ddition to normal hings, we look over a special DNA dye called SYBR green, which binds to dsDNA and then fluoresces. Itdoesn't show up if not in contact with DNA
What must you remember for real time PCR?
keep the amount in tubes constant so that there is not a fluke
Explain this graph
The red line has increased expression due to an increased concentration of the mRNA being measured. mRNA is being produced much mor in the cell that the red line was extracted from
If the clone sequence was cloned into particular plasmids calledexpression vectors, then what?
you could take advantage of the fact that it has an inducible promoter (temp or drug controlled)
- Clone the sequence in front of the promoter--> bacterial cell--> activate promoter
- makemRN--> translation
Usually a plasmid or virus designated for protein expression in cells
What is the problem with creating proteins in bacteria that are not usually produced bythem?
there may be no post translational modifications
What does epitope tagging do?
Using genetic engineering techniques, a short epitope tag is added to a protein of interest. The resulting protein contains the protein being analyzed+ the epitope tag that can be detected by antibodies. Rapid purification can occur via immunoprecipitation or affinity chromatography
How to add an epitope tag
- Use restriction enzyme to cut both gene and epitope tag
- use DNA ligate to paste together
How to purify proteins using a GST-tagged fusion protein?
- Via a pull downexperiment.
- Protein X can be fused to GST
- Column of beads with glutathione attached to it
- Specific fusion proteins will bindto the beads. Others wont
- Take them off by adding glutathione to the solution
Briefly explain the two-hybrid system.
The technique takes advantage of the modular nature of gene activator proteins (see Figure 7-42). These proteins both bind to DNA and activate transcription—activities that are often performed by two separate protein domains.
Used to look for protein-protein interactions
We know that our target proteins bind to several things but we dont know what exactly.
1) Create library in modified yeast cells (insert reporter construct binding site for DNA binding domain; plasmid (expression vector) that produces our bait
2) Fuse DNA binding domain to our bait protein
3) GEt a bunch of candidate proteins that bind to bait and modify so they have an epitope tag with TAD
extra: When this construct is introduced into yeast, the cells produce the target protein attached to this DNA-binding domain (Figure 8-51). This protein binds to the regulatory region of a reporter gene, where it serves as “bait” to fish for proteins that interact with the target protein inside a yeast cell. To prepare a set of potential binding partners, DNA encoding the activation domain of a gene activator protein is ligated to a large mixture of DNA fragments from a cDNA library. Members of this collection of genes—the “prey”—are introduced individually into yeast cells containing the bait. If the yeast cell has received a DNA clone that expresses a prey partner for the bait protein, the two halves of a transcriptional activator are united, switching on the reporter gene
- If bait and candidate bind, TAD brought to transcription point, where transcription is allowed tobegin
- If reporter gene is on, prey is the binding partner for bait
Steps to sequencing a genome?
- 1) extract genomic DNA
- 2) create a genomic library (clone for each fragment)
- 3) spread out and grow on plates-->select the clones (isolate and store them)
- 4) sequence (T3 or T7 promoter=every single clone); not in order
- 5) Use a different restriction enzyme in to get overlaps
What is the purpose of real time pcr?
used to amplify and simultaneously detect or quantify a targeted DNA molecule.
What does FRET test?
Protein protein interaction
- Fusion proteins are made attached to fluorescent proteins
- - GFP is excited by blue light and emits green light.
- - BFP is excited by violet light and emits. Luke light
An interaction leads to excitement of BFP with violet light and emittance of green light due to the closeness of the two proteins. Te blue light emitted from BFP is transferred to GFP, which emits green light
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