Universal Mouse Genotyping Protocol (downloadable pdf click here)

This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon, Transgenic Research 12, 521-522 (2003).  Primers thirty nucleotides in length are picked that will produce an amplimer from the sequence of interest. A single set of PCR conditions has been developed that will produce an amplimer from any primer set.  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 may be included in the reaction to detect multiple sequences, including amplimers that serve as internal controls.  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.

Primer Design

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 (shorter amplimers usually work better). The table shows proven primer pairs.

human α1-antitrypsin

432bp amplimer length

 

5' primer sequence:

GCA AAT GGG AGA GAC CCT TTG AAG TCA AGG

 

3' primer sequence:

CTG GCA GAC CTT CTG TCT TCA TTT TCC AGG

ApoE wild type

400bp amplimer length

 

5' primer sequence:

TAT CTA AAC AGA CTC CAC AGC CTC CAG ACC

 

3' primer sequence:

CGA AGC CAG CTT GAG TTA CAG AAT GGG ATC

ApoE knockout

600bp amplimer length

 

5' primer sequence:

GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT

 

3' primer sequence:

CGA AGC CAG CTT GAG TTA CAG AAT GGG ATC

β-galactosidase

389bp amplimer length

 

5' primer sequence:

GTT GCA GTG CAC GGC AGA TAC ACT TGC TGA

 

3' primer sequence:

GCC ACT GGT GTG GGC CAT AAT TCA ATT CGC

cre-recombinase

408bp amplimer length

 

5' primer sequence:

GCA TTA CCG GTC GAT GCA ACG AGT GAT GAG

 

3' primer sequence:

GAG TGA ACG AAC CTG GTC GAA ATC AGT GCG

Fabpi (internal standard)

194bp amplimer length

 

5' primer sequence:

TGG ACA GGA CTG GAC CTC TGC TTT CCT AGA

 

3' primer sequence:

TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG

Fabpi (internal standard)

466bp amplimer length

 

5' primer sequence:

CCT CCG GAG AGC AGC GAT TAA AAG TGT CAG

 

3' primer sequence:

TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG

firefly luciferase

323bp amplimer length

 

5' primer sequence:

AGA CGC CAA AAA CAT AAA GAA AGG CCC GGC

 

3' primer sequence:

TAT AAA TGT CGT TCG CGG GCG CAA CTG CAA

green fluorescent protein

280bp amplimer length

 

5' primer sequence:

GCA CGA CTT CTT CAA GTC CGC CAT GCC

 

3' primer sequence:

GCG GAT CTT GAA GTT CAC CTT GAT GCC

human growth hormone

360bp amplimer length

 

5' primer sequence:

CTG CAC CAG CTG GCG TTT GAC ACC TAC CAG

 

3' primer sequence:

TTT CTG TTG TGT TTC CTC CCT GTT GGA GGG

neomycin phosphotransferase

380bp amplimer length

 

5' primer sequence:

TGC TCC TGC CGA GAA AGT ATC CAT CAT GGC

 

3' primer sequence:

CGC CAA GCT CTT CAG CAA TAT CAC GGG TAG

P53 wild type

335bp amplimer length

 

5' primer sequence:

TGA GTG CTA GCT AGG CTT AGA GGT GCA AGC

 

3' primer sequence:

AGT GGA TGG TGG TAT ACT CAG AGC CGG CCT

P53 knockout

550bp amplimer length

 

5' primer sequence:

CAT CGC CTT CTA TCG CCT TCT TGA CGA GTT

 

3' primer sequence:

AGT GGA TGG TGG TAT ACT CAG AGC CGG CCT

reeler wild type

190bp amplimer length

 

5' primer sequence:

CTG CTA CAC AGT TGA CAT ACC TTA ATC TAC

 

3' primer sequence:

AGA GCC TAG AGG TTA GGG ACA CAA CTC TTC

reeler knockout

340bp amplimer length

 

5' primer sequence:

TAA GGG AGT CCT GGT CTC TTT CTG TCT TTA

 

3' primer sequence:

AGA GCC TAG AGG TTA GGG ACA CAA CTC TTC

The Fabpi pairs amplify a sequence from an endogenous murine gene and are suitable for inclusion in any reaction as internal controls.  he two Fabpi sets produce amplimers of different lengths to allow inclusion one set with mutation-specific primers that produce amplimers of any length.  The Fabpi primers work with the other primers shown in the table.

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 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 100pgµL 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, you need to be able to see a band at the 0.01pg level.  Anything less sensitive may allow multiple copy detection, but it may be to your advantage to test a different set of primers.

PCR reaction

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°C, except for the betaine which is kept at room temperature and the PCR buffer which is stored at 4°C.

Reagent Stock Solutions

water

 

4M betaine in water

Sigma B2754

10mM cresol red in water

Sigma C9877

20mM dNTP mix in water

100µM dATP Promega U120A
100µM dGTP Promega U121A
100µM dCTP Promega U122A
100µM dTTP Promega U123A

10X KLA Buffer pH = 9.2

Wayne Barnes 314.362.3351
barnes@barnes1.wustl.edu

Klentaq LA (DNA polymerase)

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

 

Reaction Mixture

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.

water

5.1 µL1

1Adjust water so that reaction contains 20µL after adding DNA template.

betaine

6.5 µL

 

10X buffer

2.0 µL

 

dNTP mix

0.05 µL

 

cresol Red

0.10 µL

 

5' Fabpi primer

0.12 µL2

2Fabpi primers work best at this concentration - 1.0µL should be used for all other primers.

3' Fabpi primer

0.12 µL2

5' transgene primer

1.0 µL

 

3' transgene primer

1.0 µL

 

Klentaq LA

0.04 µL

 

mouse DNA sample

4.0 µL

 

 

 

PCR cycling

Run the following program:

After the PCR reaction is finished, electropherese each entire PCR sample through a 2% agarose gel at 120V for approximately one hour.

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°C.  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.

Reagent Stock Solutions

base solution 50X stock = 1.25M NaOH, 10mM EDTA pH=12

·         250 mL 5N NaOH

·         20 mL 0.5M EDTA

·         bring volume to 1L with water

·         Adjust pH to 12.0 with NaOH/HCl

base solution 1X working stock = 25mM NaOH, 0.2mM EDTA pH=12

·         add 1mL of 50X stock solution to 49mL water

neutralization solution 50X stock = 2M Tris-HCl pH=5

·         315.2g Tris HCl

·         bring volume to 1L with water

·         adjust pH to 5.0 with NaOH/HCl

neutralization solution 1X working stock = 40mM Tris-HCl pH=5

·         add 1mL of 50X stock and 49mL water

Protocol