Restriction Mapping Of pGLO Flashcards
(10 cards)
Experimental procedure
- Set up restriction enzyme digests of DNA samples (A-G) with 3 restriction enzymes = EcoRI, EcoRV and Nhel (single, double & triple digests)
- Incubate at 37 degrees for 45 minutes
- Add loading buffer (blue LB) to each digest and load samples on agarose gel together with molecular weight markers (M)
- Run the agarose gel at 100V until the blue dye (BPB) is 2-3 cm from the end of the gel
- Photograph the gel under UV light to reveal the DNA bands
How is the calibration curve made?
LogMW is the x-axis and distance migrated is the y-axis
This is plotted using MW markers DNA fragments of known size and should be a linear relationship
What is restriction mapping?
Plan diagrams of the plasmid that show the relative positions of the cutting sites of different restriction enzymes
These maps can be used to distinguish different plasmids and are often employed when constructing recombinant DNA molecules to track insertion or deletion of sequences
What are bacteria vulnerable to infection by viruses called?
Bacteriophages
What are endonucleases?
Cut and degrade any foreign DNA including that of an attacking phage
They are termed restriction enzymes because they restrict infection by bacteriophages
They cut DNA at a specific point WITHIN a specific sequence of base pairs and this site is known as the recognition sequence - different endonucleases have different recognition sequences
Describe recognition sequences in bacteria
The recognition sequence for a bacterial restriction endonuclease may also be present in the bacterial DNA so bacteria chemically modify nucleotides within the recognition sequences in their own genome (by methylation), making them ‘invisible’ to their own endonucleases
Each particular type of bacterium has a restriction enzyme (or several different ones) that cuts a specific DNA sequence and is paired with a methyl-transferase enzyme that protects this same sequence in the bacterial genome
Type I and type III restriction enzymes have both endonuclease and modification methylase activities on the same protein whilst, type II enzymes have the 2 activities on separate enzymes so can be used simply to cleave DNA at specific sites
How is a restriction map constructed?
- A number of restriction enzymes that cut the plasmid DNA at different sites are used
- Each enzyme cuts the plasmid DNA at all of the recognition sites for that enzyme, acting as a pair of molecular scissors
- The number of fragments depends on the number of cuts (sites) and the size/length of the fragments depends on the location of the sites, sites that are relatively close to each other will produce smaller fragments
- Fragments and their size can be determined agarose gel electrophoresis
- A map can then be assembled by comparison of the lengths of the fragments from at least 2 single and one double digest
What are DNA ladders?
Molecular size markers = fragments of known size
Allow you to calibrate the gel
How to analyse data using the photograph
- measure how far each DNA band has migrated from the wells (in mm) using a ruler
- plot graph using this data and the MW is the size of the fragments in base pairs (should be linear)
- measure distance from the middle of the loading well to the centre of each DNA band
- use standard curve to calculate the size of the DNA fragments produced by the various restriction enzymes
Key things to draw a restriction map
- Determine the overall size of the plasmid in kbp
- Work out how many times each enzyme cuts the plasmid
- Decide arbitrarily where the EcoRI cutting site is (e.g. the 12 o’clock position)
- Use the double digest data to decide where EcoRV cuts in relation to EcoRI
- Use the other double digest data to decide where Nhel cuts in relation to the other two sites
- Go back and check the triple digest and make sure the answer fits all the data presented
