Chapter 4 Flashcards
Four basic steps for cloning
1) Prepare Vector and Insert DNA as necessary. Isolate/purify/amplify DNA (e/g/, mini-preps, PCR). Treat DNA as necessary (e.g., restriction digests, modifications).
2) Mix and Ligate vector and insert DNA
3) Transform DNA into bacteria
4) Select/Screen colonies for desired clone
Taq DNA polymerase
Has some terminal deoxynucleotidyl transferase activity. This results in Taq polymerase adding an untemplated nucleotide to the 3’ ends of the DNA - this is typically an A.
Other DNA polymerases with 3’ –> 5 exonuclease activity do not add an extra A to the ends of the PCR product
TOPO TA cloning
Takes advantage of the extra A added by Taq DNA polymerase. This cloning mechanism does not require DNA ligase, because a topoisomerase actually catalyzes phosphodiester bond formation
Designing primers containing restriction sites
1) Identify potential primers to amplify region
2) Add on bases to generate desired restriction site(s)
3) Denature DNA and anneal primers
4) Extension of DNA synthesis from primers
5) After multiple rounds of denaturation, annealing, and extension
6) PCR product can now be digested with EcoRI and KpnI
Phosphatase treatment of DNA
When a circular vector is cleaved with a single enzyme (regardless of type of overhang) the ends can easily religate back together. To avoid recircularization, the digested vector ends can be treated with phosphatase. Phosphatase removes the 5’P groups and therefore cannot be used to ligate back together. However, if an insert containing 5’P groups is present, two phosphodiester bonds can form. Once transformed into bacteria, the cell will add 5’ P to remaining ends, and complete the ligation
Polynucleotide kinase treatment of DNA
Adds P groups to the 5’ ends and requires ATP. This can be used to replace the 5’ P with a radioactively labeled P
Gel electrophoresis
Negative charged DNA (PO4 groups) means DNA runs toward the positive electrode (anode). DNA has the same mass/charge ratio, no matter what length
Make a 1% (w/v) gel
1) 1g of agarose in 100 mL of buffer.
2) heat to above 60 C to dissolve
3) Pour the gel into the tray with comb
4) Gel forms at 30 C
5) Gel comb is removed, box filled with buffer
6) Samples mixed with tracking dye
7) Samples loaded into wells
8) Electric current is applied
9) Gel is run until DNA is separated as desired
Tracking dye purposes
1) Typically contain glycerol, sucrose, or ficoll to weigh down DNA
2) Typically contain dye to track migration of DNA indirectly
Common dyes
A) Xylene cyanol - aqua blue color; 3,000-4,000 bp
B) Bromophenol blue - dark blue/purple color; 400-600 bp
C) Orange G - orange color; 50-100 bp
Ethidium bromide
Is and intercalating agent, is a mutagen, and is fluorescent under UV light when bound to DNA.
EtBr intercalates between A-T basepairs
Polyacrylamide gel electrophoresis
Acrylamide is a potent neurotoxin
Polyacrylamide is no longer toxic
Polyacrylamide gel electrophoresis can provide 1 bp resolution
Agarose properties
Non-toxic
100-50,000 bp
0.5-2.0% in gel
Easy preparation
Polyacrylamide properties
Toxic when unpolymerized
1-500 bp
3.5-20% in gel
Tedious preparation
Uses for DNA gel electrophoresis
1) Quantification of DNA - intensity fo each band is indicative of the amount of DNA present
2) Isolation of DNA - DNA fragments can be separated from other DNA fragments by size. The desired DNA fragment can then be extracted from the gel
3) Restriction mapping - analyzing DNA by examining restriction digested DNA, either for identification or mapping purposes
4) Sequencing analysis - used to determine the sequence of DNA
