This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon (Transgenic Res. 12, 521-522 (2003)).For those familiar with PCR genotyping, this method differs from the typical protocol by utilizing a unique enzyme (Klentaq), 30mer primers, and a 68° annealing temperature.
PCR Amplification is used to genotype mice by detection of sequences not normally present in the murine genome. Primers binding to a site unique to a mutation can be used to amplify a segment of DNA, which can be detected with agarose gel electrophoresis. For example, primers can be chosen from the sequence for bacterial neomycin phosphotransferase typically used in knockout targeting vectors. An amplimer will be produced when this sequence is present but not when it is absent.
A set of PCR conditions has been developed that will produce a detectable amplimer with any primers from sequences present in a single copy in the murine genome. The only criteria are that primers must contain approximately 50% guanosine or cytosine, and that the amplimer be 100-400 nucleotides in length. More than one primer set is normally included in the reaction so that a band will be produced in each reaction to serve as a control. For example, a transgene may be detected by primers that bind to LacZ sequences contained in the transgene, but primers to a normal mouse gene will also be included. A band will be detected for the endogenous mouse gene in all samples, and transgenic mice will be indicated by the presence of an additional band. The detection of a band in all samples serves to control for DNA quality and correct amplification. Positive controls (DNA with each possible genotype) and a control with no DNA present should be included in each reaction set.
DNA is prepared by briefly boiling a mouse toe or other tissue in base, then adding a neutralization solution [Biotechniques 29(1), 52-54 (2000)], as described below. This method produces DNA which performs well in PCR assays, and is stable for up to a month at -20°
Primers must be picked that will amplify the sequence of interest but no other sequence. Primers should be thirty nucleotides in length and contain 40-60% total guanosine or cytosine residues. The primers should not have homologous three prime ends, repetitive sequences, or long runs of a single residue. The amplimer should be 100-400 nucleotides in length, where shorter amplimers usually work better. We have developed suitable primer sets for many common sequences in induced mutations, as shown on our primer set page. The PCR assay should include at least two primer pairs so that each reaction is expected to produce at least one amplimer in all samples. Internal control primers from an endogenous mouse gene have been developed that will produce amplimers of 200 base pairs or 500 base pairs.
Sensitivity: This PCR genotyping assay has proven sensitive enough to detect genomic sequences present as a single copy at one allele. This sensitivity is obviously necessary for induced mutations such as gene knockouts, but is also important in identifying transgenic founder animals that may be present as a single copy. We have compared the sensitivity of the assay between plasmid standards and murine genomic DNA containing so that assay sensitivity may be tested before generating mice containing the mutation. To test sensitivity, make serial dilutions of your plasmid containing the mutation in water containing mouse genomic DNA at a concentration of 500ng/µL. Plasmid concentrations should be tenfold dilutions spanning 100pg/µL to 0.01pg/µL. Set up the standard PCR mix, using 1µl of each dilution as your DNA template. In order to assure detection of single copy integrated transgenes or induced mutations, you need to be able to see a band at the 0.01pg level.
Stock solutions are prepared ahead of time, and then used to assemble the working master mixture. The master mixture is stable for at least two hours at room temperature after all ingredients are added. The stock solutions can be made ahead of time and stored at ‑20°, except for the betaine, which is kept at room temperature, and the PCR buffer, which is stored at 4°.
|4M betaine in water||Sigma B2754|
|10mM cresol red in water||Sigma 11,448-0|
|20mM (each) dNTP mix in water||dATP Promega U120A|
|dGTP Promega U121A|
|dCTP Promega U122A|
|dTTP Promega U123A|
|10X KLA Buffer pH = 9.2||Wayne Barnes 314.362.3351|
|Klentaq LA (DNA polymerase)||firstname.lastname@example.org|
|20μM 5'-internal control primer in water|
|20μM 3'-internal control primer in water|
|20μM 5'-transgene primer in water|
|20μM 3'-transgene primer in water|
The total reaction volume is 20µl including the DNA sample, and cycling is done in thin-walled tubes in a machine with a heated lid. Note that this reaction mixture contains four primers, two of which are Fabpi internal control primers.
1. Make a master mix containing the following reagents, where volumes listed are for one reaction. You will need a master mix with enough reagents for the number of samples and controls to be run, plus 10% extra for losses during pipeting.
Adjust water so that reaction contains 20µL after adding DNA template.
Fabpi primers work best at this concentration - 0.5µL should be used for all other primers.
|5' Fabpi primer||0.25µl2|
|3' Fabpi primer||0.25µl2|
|5' transgene primer||1µl|
|3' transgene primer||1µl|
Mix master mix well by flicking tube gently, then pulse spin in a microfuge.
Aliquot 16.0µl of master mix into thin walled PCR tubes.
Add 4µL of DNA sample to each tube.
Place tubes in PCR machine.
Run the following program:
step 1=93° for 1 minute
step 2=93º for 20 seconds
step 3=68°for 3 minutes
repeat steps 2 and 3 for a total of 30 cycles
step 4=4° until samples are retrieved
After the PCR reaction is finished, electrophorese each PCR sample through a 2% agarose gel at 120V for approximately one hour in TAE or TBE buffer.
HotSHOT Preparation of Murine DNA for PCR
This procedure is adapted from Biotechniques 29(1), 52-54 (2000). Briefly, a single toe is clipped from a mouse aged five days through adult. The toe is placed in a PCR tube within one hour of cutting and may be stored frozen at ‑20° for extended periods. DNA is released from the tissue by heating in a base solution, then adding a neutralization solution. The working stocks can be made from the concentrate and used for two weeks. Stock solutions should be replaced every six months.
BASE SOLUTION: A 50X stock is prepared at 1.25M NaOH, 10mM EDTA, which should be around pH=12. Mix together 250mL 5N NaOH, OR 50g of NaOH crystals and 20mL 0.5M EDTA, and water to 1000mL total volume. Prepare a working 1X stock (25mM NaOH, 0.2mM EDTA) by combining 1mL 50X stock with 49mL water.
NEUTRALIZATION SOLUTION: A 50X stock is prepared at 2M TRIS-HCl, pH=5.0. Dissolve 315.2g TRIS HCl in 1000mL water. Prepare a 1X working stock (40mM Tris-HCl pH=5.0) by combining 1mL 50X stock with 49mL water.
Cut toe and place in a thin-walled PCR tube, and freeze until ready to elute DNA. Toes may be kept at room temperature for up to an hour before freezing, but should be thawed by adding the base solution and heated immediately.
Add 75µL of base solution 1X working stock to each sample.
Place tubes at 95° in a PCR machine (or bath or block) for 30 minutes.
Cool to room temperature or below.
Add 75µL neutralization solution 1X working stock and vortex.
Use 2µL of this mixture in a 10µL PCR reaction.