MHC haplotyping for cynomolgus, rhesus, and pig-tailed macaques using the UW-Madison 16 microsatellite marker panel

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MHC haplotyping for cynomolgus, rhesus, and pig-tailed macaques using the UW-Madison 16 microsatellite marker panel

1. Genomic DNA isolation from whole blood or cells

To facilitate rapid genomic DNA isolation from fresh whole blood or frozen cell samples from up to 32 animals simultaneously, we use a MagNA Pure robot and MagNA Pure LC DNA isolation kit according to instructions provided by Roche Applied Science (https://www.roche-applied-science.com). Other DNA isolation techniques such as QIAamp DNA Blood Mini kits or DNeasy Blood and Tissue Kits from Qiagen (http://www.qiagen.com) can be substituted if this robot is not available. Regardless of isolation technique, proper protective equipment should be worn when handling nonhuman primate samples and DNA isolation reagents. Typical DNA concentrations from 300ul blood samples with the MagNA Pure LC DNA Isolation Kit range between 10–35 ng/ulin an elution volume of 100 ul.

 

(a) Ensure that the MagNA Pure LC Instrument has the proper DNA purification protocols installed as detailed by the MagNA Pure LC DNA Isolation Kit manual.

(b) Prepare the isolation kit reagents as detailed in the manual (https://www.roche-applied-science.com).

(c) Aliquot 300 ul of whole blood or white blood cells, or ~1 x 106 peripheral blood mononuclear or cultured cells/100 ul as directed by the manual.

(d) Set up the instrument using the appropriate protocol for the starting material. Instructions supplied by the software will calculate individual reagent volumes based upon number of samples to be isolated.

(e) Upon completion of the isolation run, quantitate DNA concentrations with a NanoDropTM (Thermo Scientific) normalize DNA samples to 10 ng/ul prior to proceeding with microsatellite amplification.  If necessary, DNA samples can be stored indefinitely at -80 °C prior to PCR.

2.  Preparation of fluorescently labeled microsatellite primer stocks (6 multiplex mixes)

Multiplex Mixes A – F were developed for 16 microsatellite loci spanning the 5Mb MHC region of macaques (see Table 1. UW-Madison 16 Microsatellite Marker Panel for MHC Genotyping).  Prepare each mix as specified in table below (yields 1ml working primer stocks):

 

 

Marker

Dye

 Master stock [uM] F/R-primers

Final target [uM]

uL F  -primer

uL R-primer

ul dH2O

Mix A

D6S2970

FAM

100/25

0.3

7.5

30

 

 

D6S2876

FAM

25/25

0.3

30

30

 

 

D6S2745

HEX

100/25

0.3

7.5

30

 

 

 

 

 

 

 

 

865

Mix B

D6S2704

FAM

100/25

0.075

1.9

7.6

 

 

MICA

TAMRA

100/100

0.15

3.8

3.8

 

 

PO3-193435

HEX

100/100

0.3

7.5

7.5

 

 

 

 

 

 

 

 

968

Mix C

D6S2892

FAM

100/25

0.3

7.5

30

 

 

D6S2691

TAMRA

100/25

0.3

7.5

30

 

 

D6S2747

HEX

100/25

0.15

3.8

15

 

 

 

 

 

 

 

 

906

Mix D

D6S2972

FAM

100/100

0.3

7.5

7.5

 

 

DRA-CA

FAM

100/100

0.3

7.5

7.5

 

 

 

 

 

 

 

 

970

Mix E

D6S2669

FAM

100/25

0.037

0.93

3.7

 

 

D6S2782

HEX

100/25

0.037

0.93

3.7

 

 

D6S2793

HEX

100/25

0.037

0.93

3.7

 

 

 

 

 

 

 

 

986

Mix F

DO5-144699

FAM

100/100

0.3

7.5

7.5

 

 

DO5-104184

HEX

100/100

0.3

7.5

7.5

 

 

 

 

 

 

 

 

970

3.  PCR amplification of normalized genomic DNA

(a) Prepare 6 PCR master mixes (A-F) for N number of samples:

N x 5 ul of 2X Phusion HF DNA polymerase Master Mix (New England Biolabs)

N x 4 ul multiplex primer mix (A-F) prepared in step 2. 

 

(b) Combine 9 ul of PCR master mix with 1 ul of normalized DNA (10 ng)/reaction. 

(c) Amplify 10 ul microsatellite PCR reactions using the following program with a Tetrad thermocycler (Bio-Rad Laboratories):

 

Initial denaturation of 98 °C for 30 sec

 

98 °C for 5 sec

64 °C for 5 sec           2 cycles

72 °C for 20 sec

 

98 °C for 5 sec

62 °C for 5 sec           2 cycles

72 °C for 20 sec

 

98 °C for 5 sec

60 °C for 5 sec           2 cycles

72 °C for 20 sec

 

98 °C for 5 sec

58 °C for 5 sec           4 cycles

72 °C for 20 sec

 

98 °C for 5 sec,

50 °C for 5 sec,          24 cycles

72 °C for 20 sec

 

Final elongation of 72 °C for 5 min

4.  Product Size Analysis using Capillary Electrophoresis

Capillary electrophoresis is used to determine size of the fluorescently labeled products generated in step 3 with a 3730xl Genetic Analyzer (Applied Biosystems).

 

(a) Prepare size standard mix for N number of samples:

N x 8.6 ul Hi-DiTM formamide (Applied Biosystems) plus 0.4 ul MegaBACETM ET550 Rox size standard (GE Healthcare Life Sciences)

 

(b) Dilute 1 ul of PCR product into 9 ul of size standard mix in each well.

(c) Denature PCR products at 98 °C for 2 min before loading plate onto the 3730xl Genetic Analyzer and beginning run with Foundation Data Collection program (Applied Biosystems).

(d) Microsatellite peak sizes are scored using DAx Acquisition and Data Analysis software (Van Mierlo Software Consultancy; www.dax.nl). 

Note: Due to the limited genetic diversity in the MHC region of Mauritian cynomolgus macaques, microsatellite peak values for each marker profile can be assigned to 1 of 7 common haplotypes (see Figure 1: Known Mauritian Cynomologus Macaque Haplotypes and Associated MHC Alleles).  Additional pedigree analysis is required to assign individual microsatellite markers to specific MHC haplotypes of other nonhuman primates.

 

 

 

 

 

 

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