Question: The lab has introduced new mice. How do we determine their genotype?
 

Answer: Common methods for mouse genotyping include conventional PCR, qPCR, and sequencing. Let's discuss them in detail.

Genotyping is also necessary in the following situations besides newly introduced mice:

1.  Before Breeding Pairing: When breeding mice, genotyping the breeding pair ensures the parent genotypes are accurate. This effectively prevents confusion in offspring genotypes due to parental genotype errors, thereby improving breeding efficiency and the reliability of experimental results.

2.  Offspring Mice: If the parents produce offspring with various genotypes (e.g., heterozygous x heterozygous), the offspring need to be genotyped. This allows for the selection of mice with the specific genotype required for the experiment, ensuring its smooth progress.

 

01. Common Genotyping Methods
 

1.  Conventional PCR Genotyping

The most commonly used genotyping method currently. It uses specific primers and PCR technology to amplify the target gene fragment. The genotype is then determined by analyzing the band differences of the PCR products via agarose gel electrophoresis.

a.  Characteristics: Commonly used in oncology, immunology, and inflammation research. Simple to operate, low cost, suitable for most laboratories.

 

2.  qPCR Genotyping
 

qPCR (Real-time Quantitative PCR) is a more precise genotyping method. It monitors the PCR amplification process in real-time via fluorescent signals, allowing not only genotype determination but also quantitative analysis of gene expression levels.

a.  Characteristics: High sensitivity, strong specificity, but requires higher-level equipment and technical expertise.

3.  Sequencing

For complex genotyping, such as point mutation mice, sequencing is an essential tool. It directly reads the base sequence of the target gene, allowing for accurate genotype determination.

a.  Characteristics: Higher cost, but provides the most accurate and reliable results.

 

Question: The most common method is conventional PCR genotyping. What are the operational steps for this method?
 

02. PCR Genotyping Operational Steps
 

I. Sample Collection:

Common sample sources include mouse tail tips, ear notches, and toe tissue. Sampling is generally recommended 9-14 days after birth. Samples can be stored at -20°C for later use.

II. DNA Template Extraction:

2.1 Reagents

a.  Mouse Tail Lysis Buffer

b. Proteinase K Solution: Proteinase K (20mg/ml in MilliQ water), stored at -20°C.

c.  TE Buffer
 

2.2 Experimental Procedure
 

1.  Add 300 µl of lysis buffer and 5 µl of Proteinase K (20 mg/ml) to each sample. Digest at 55°C overnight (not less than 4 hours).

2.  Ethanol Precipitation: Add 600 µl (double volume) of -20°C pre-chilled absolute ethanol. Invert the tube until flocculent precipitate is clearly visible.

3.  Centrifuge at 12,000 rpm for 5 minutes.

4.  Drying: After centrifugation, pour off the supernatant. Place the tube upside down on a flat piece of paper (ensure it is upright so the liquid can flow to the tube opening). After about 5-10 minutes, reposition the upright tube and gently press it downwards (to absorb liquid accumulated at the opening with the paper, saving drying time). Let it dry completely for 30-60 minutes until the precipitate becomes transparent, indicating good quality extracted DNA.

5.  Add 200 µl of MilliQ water or TE solution. Dissolve the DNA thoroughly by inverting and shaking the tube.

6.  Use 3 µl of gDNA template for PCR in a 25 µl reaction system.
 

III. PCR:

3.1 Reaction System (25 µl system)

3.2 Reaction Conditions (Touchdown PCR)

IV. Electrophoresis:
 

4.1 Gel Preparation: Prepare a 1-3% agarose gel based on the target band size. Dissolve 1g-3g agarose in 100 ml TAE buffer, heat in a microwave until melted. Add 10 µl nucleic acid dye . Pour into the gel tray, insert a comb, and let solidify.

4.2 Loading: Load an appropriate volume of PCR product onto the agarose gel . Include a suitable DNA ladder/marker for size reference.

4.3 Running Gel: Run the gel at 120V for 20-30 minutes.

4.4 Imaging: Observe and photograph the gel using a gel imaging system.

4.5 Analysis: Edit the gel image and generate a genotyping results report.

 

Answer: The following points usually require attention during the experiment.
 

03. Important Considerations
 

1.  Control Setup: Setting appropriate controls is crucial in genotyping experiments. Typically needed:

a.  Wild-type Control: To assess the genotyping assay and whether the PCR system is suitable.

b.  No-template Control (NTC): Contains no tissue/DNA. Controls for PCR system contamination.

c.  Internal Positive Control: Primers usually amplify a genomic DNA region unrelated to the target gene. Assesses the suitability of the PCR system and template quality.

2.  Primer Design: Key to successful PCR. Considerations include primer GC content, secondary structures, dimer formation, annealing temperature, length, and specificity. Poor primer design can lead to low PCR yield or absence of the target band.

3.  Contamination Control: Strictly control contamination during the experiment. Avoid cross-contamination during sample collection. Perform DNA extraction and PCR setup in a sterile environment using disposable consumables and nuclease-free reagents.

 

Question: What are some common problems in PCR genotyping experiments?
 

04. Common Problems and Solutions
 

1.  Low or No Target PCR Product:

a.  Possible Causes: PCR inhibitors in the tissue extract, insufficient tissue digestion, poor primer design, high GC content or long length of the amplicon.

b.  Solutions: Test for inhibitors, extend digestion time, optimize primer design, use reagents and conditions suitable for high-GC or long-fragment amplification.

2.  Non-specific Bands:

a.  Possible Causes: Suboptimal annealing temperature, primer-dimer formation, excessive template amount.

b.  Solutions: Optimize annealing temperature, reduce template amount.

3.  Smearing/Diffuse Bands:

a.  Possible Causes: Degraded DNA template or suboptimal PCR conditions.

b.  Solutions: Optimize DNA extraction to avoid degradation; adjust PCR conditions (e.g., denaturation time, annealing temperature).

 

References:

[1] Relevant WeChat article on genotyping.

[2] Wang Tinghua. (2005). PCR Theory and Techniques (Third Edition). Science Press.

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