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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Methods Mol Biol. Author manuscript; available in PMC 2012 October 5.
Published in final edited form as:
PMCID: PMC3464904

KIR Genotyping by Multiplex PCR-SSP


Diversity across KIR haplotypes stems from differences in numbers of inhibitory and activating receptors, as well as allelic polymorphism of individual genes. The KIR locus has undergone large expansions and contractions over time and is believed to be coevolving with genes encoding its HLA class I ligands located within the MHC locus. KIR and HLA compound genotypes have been associated with susceptibility to or protection from infectious, autoimmune, reproductive, and malignant disorders. We describe here a simple and reliable multiplex PCR-SSP (sequence-specific priming) method for relatively rapid and inexpensive genotyping of 15 KIR genes using standard agarose gel electrophoresis.

Keywords: KIR genotyping, NK cell, KIR haplotypes, multiplex PCR, PCR-SSP

1. Introduction

Natural killer (NK) cells are central to the innate immune system and they represent the first line of defense against bacteria, parasites, viruses, and malignant cells (1). NK cells comprise 5–15% of circulating lymphocytes and are also found in tissues including the liver, peritoneal cavity, and placenta. They can mediate spontaneous killing of infected or transformed target cells and produce immunoregulatory cytokines and chemokines that stimulate the adaptive immune response (2, 3). In addition, they engage in interactions at the maternal–fetal interface that are critical for successful pregnancy (4).

NK cell activity is tightly controlled by activating and inhibitory interactions through numerous cell surface receptors. They respond to targets that express aberrant levels of MHC class I, and this involvement of MHC class I molecules in NK cell recognition of self vs nonself was originally described over two decades ago (5). MHC class I molecules are the essential signature of self that NK cell inhibitory receptors engage in order to avoid NK cell attack of normal cells (57). The original ‘missing self’ hypothesis (8) proposed that downregulation of class I expression on target cells leads to spontaneous destruction by NK cells. Current knowledge of NK cell biology suggests that NK cells not only patrol for abnormal cells that lack MHC class I but also for those that over-express ligands for activating receptors such as altered MHC (‘altered self’) and non-MHC ligands (‘induced self’ or ‘nonself’) (9).

The killer cell immunoglobulin-like receptors (KIR) are one of the main types of MHC class I-specific receptors utilized by human NK cells, and they are comprised of both activating and inhibitory counterparts that are encoded by highly homologous sequences. The KIR locus maps to chromosome 19q13.4 within the leukocyte receptor complex (LRC) (10). The high sequence similarity of the KIR genes may facilitate the occurrence of non-allelic homologous recombination (NAHR) (11, 12) and probably partially explains expansion and contraction of KIR haplotypes (10). KIR haplotypes have been broadly classified into two groups referred to as A and B, both of which are found in most populations but sometimes at very different frequencies. Haplotype A is invariant in terms of gene content while haplotype B is quite variable (13) (Fig. 25.1). Currently, more than 40 different B haplotypes based on gene content have been described [summarized in (14)], and further diversity is afforded by allelic polymorphism of individual KIR genes such that it is unlikely that any two randomly selected individuals have identical KIR genotypes (15). The highly polymorphic and genetically unlinked KIR and MHC class I loci appear to have coevolved to give selective advantage to higher order mammals with respect to reproduction and resistance against infections. Thus, it is not surprising that an ever-increasing number of genetic studies have shown that combinations of KIR and HLA confer either protection against or susceptibility to infectious, reproductive, autoimmune, and malignant disorders (16). KIR genotypic variation across populations is also important from an evolutionary standpoint since differences may in part reflect a response to differences in exposure to infectious agents across populations (17).

Fig. 25.1
The leukocyte receptor complex on chromosome 19q13.4. The KIR gene cluster is located within the leukocyte receptor complex. The KIR locus is expanded to show the order of the genes on chromosome 19. Haplotypes A and B are labeled as such. The A haplotype ...

This chapter will describe a method for multiplex KIR genotyping based on our previously published methodology (18). We have used the same primers but in multiplex combinations that can be resolved by gel electrophoresis. This method detects the presence/absence of KIR genes, thus providing KIR gene profiles.

2. Materials

2.1. PCR Amplification

  1. Good-quality DNA from tissues or cells isolated using standard protocols at a concentration of 50–100 ng/µl (see Notes 1 and 2).
  2. Oligonucleotide primer mixes containing forward and reverse primers (see Table 25.1 for primer sequences and Table 25.2 for list of primer mixes and primer concentrations).
    Table 25.1
    KIR genotyping primers
    Table 25.2
    Primer mixes for multiplex PCR
  3. 50 mM MgCl2.
  4. dNTP mix: 25 mM each of dATP, dTTP, dGTP, and dCTP.
  5. 10× PCR buffer: 200 mM Tris–HCl, pH 8.4, and 500 mM KCl.
  6. Taq DNA polymerase (see Note 4).
  7. 384-Well PCR plates. Each sample will require 12 wells, thus allowing genotyping of 32 samples per plate.
  8. Multichannel pipettors (8 and 16 channels).

2.2. Gel Electrophoresis

  1. Orange G loading buffer: 0.5% Orange G, 20% Ficoll, and 100 mM ethylenediaminetetraacetic acid (EDTA).
  2. 10× TAE electrophoresis buffer: 400 mM Tris, 200 mM acetic acid, and 10 mM EDTA.
  3. 100 bp DNA ladder.
  4. Electrophoresis grade agarose.
  5. Horizontal electrophoresis chamber.
  6. Gel casting tray with 50-well combs having teeth appropriately spaced to allow for loading with a multichannel pipettor. The Centipede™ horizontal model D3–D14 wide gel system (Woburn, MA) will accommodate two combs per gel allowing for electrophoresis of eight DNA samples, each with 12 PCR reactions.
  7. 10 mg/ml ethidium bromide solution.
  8. High-voltage power supply.
  9. Photographic means of gel documentation (ultraviolet light source, camera).

3. Methods (KIR Genotyping by Gel Electrophoresis)

3.1. PCR

  1. Using a multichannel pipette, dispense 1 µl of each primer mix into separate wells of the 384-well PCR plate. Each sample will require 12 wells in a horizontal row (e.g., A1–A12) such that 32 samples can be accommodated on one plate.
  2. Prepare PCR cocktail of 60 µl (enough for 15 wells) for one sample as follows: 150–200 ng DNA, 7.5 µl 10× PCR buffer, 2.25 µl MgCl2 (final PCR concentration 1.5 mM), 0.6 µl dNTP (final PCR concentration 200 µM), and 0.375 µl Platinum taq polymerase. Add water to make up the volume to 60 µl. Mix well by vortexing at low speed.
  3. Dispense 4 µl of PCR cocktail for each DNA sample to each primer mix in the plate for a total PCR reaction volume of 5 µl.
  4. Cover the plate with acetate film and centrifuge briefly to pull the contents of the well to the bottom of the plate.
  5. The amplification is carried out in a programmable PCR thermal cycler with heated lid to minimize evaporation using the following program: 3 min at 94°C, 5 cycles of 15 s at 94°C, 15 s at 65°C, 30 s at 72°C; 21 cycles of 15 s at 94°C, 15 s at 60°C, 30 s at 72°C; 4 cycles of 15 s at 94°C, 1 min at 55°C, 2 min at 72°C followed by a final 7 min extension step at 72°C.

3.2. Agarose Gel Electrophoresis

  1. For one gel, prepare 150 ml of 3% w/v agarose in 1× TAE by heating until the agarose is completely solubilized.
  2. Cool the mixture to 65°C and add 5 µl ethidium bromide and gently mix to avoid bubble formation.
  3. Pour the gel mixture into the gel casting tray and insert the 50-well combs. Allow the gel to solidify (~20 min).
  4. Submerge the solidified gel in the electrophoresis chamber filled with 800 ml of 1× TAE buffer. Remove the combs.
  5. Add 5 µl of Orange G loading buffer to each PCR product and centrifuge briefly.
  6. Load 2 µl of the 100 bp DNA ladder to the first and last well of each row of wells.
  7. Using a 16-channel pipettor, load 10 µl of each PCR product into the gel.
  8. Electrophorese for 70 min at 100 V or until the Orange G has migrated 6 cm.
  9. Visualize the gel using a UV light source and photograph the gel for a permanent record (Fig. 25.2).
    Fig. 25.2
    KIR genotyping by multiplex PCR-SSP and agarose gel electrophoresis. (A) Agarose gel electrophoresis depicting the presence of all the KIR genes tested. (B) Gel electrophoresis results from an individual homozygous for haplotype A. This individual possesses ...

3.3. Interpretation of Result

Determination of KIR genotype using this method depends on presence or absence of detectable amplification product for each gene-specific primer pair (see Notes 911). It is important to have a good quality gel picture for accurate judgment of the product sizes, as each well contains multiple products of varying size. The most important product is the positive internal control amplicon, which should be present in all the samples. Each KIR gene is amplified using two pairs of specific primers and products should be detected for both sets of primers. If the amplification by the two gene-specific primer pairs is discrepant for any gene, the reaction should be repeated. Detectable bands as well as absence of product can be recorded in a spreadsheet such as Microsoft Excel. Some alleles of the KIR2DS4 gene have a 22 bp deletion in exon 5 that can be detected by the KIR2DS4 primers in primer mix 8. The full-length product is 219 bp while the deletion variant is 197 bp (see Fig. 25.2B).


This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.


1The quality of DNA is the most important requirement for reliable PCR-SSP results. DNA extracted from heparinized blood should be avoided, as heparin is a PCR inhibitor (19).

2A total of 100–200 ng (gel electrophoresis method) DNA is required for reliable amplification using the primer mixes described here. Increasing the number of cycles is likely to increase false-positive reactions. Therefore, if the amplification is not satisfactory because of DNA quality, it is recommended that each primer mix be amplified individually, as described in (18).

3All primer mixes should be tested with positive and negative controls when new primer mixes are made from the stock primers.

4Platinum Taq (Invitrogen) polymerase gives the most reliable and consistent results for this protocol.

5A fast ramping thermal cycler with a heated lid is recommended for best results. The ‘step-down’ program was designed for this protocol to increase specificity of the amplifications. It might be necessary to alter the PCR conditions slightly depending on the PCR machine being used.

6PCR machines should be checked regularly to ensure that all wells are amplifying uniformly. It is also important to ensure that the PCR plates fit snugly into the machine.

7The quality of dNTPs is important for reliable amplifications. Avoid repeated freeze–thawing; instead freeze in small aliquots after reconstitution.

8The highly homologous sequences of the KIR loci can lead to nonspecific amplification especially with increased numbers of cycles (>30) used for PCR.

9Some primers miss a couple of alleles of a given gene as noted in Table 25.1 and others amplify one allele of another gene. Furthermore, there are likely to be yet undiscovered alleles of a gene that might not amplify with the current set of primers and alternatively some of these might cross-react with alleles of other KIR genes. We strongly recommend routine checks of the KIR database ( for new alleles which might require designing new primers. It should also be remembered that most of the available KIR gene sequences have been obtained from Caucasians and thus may not include alleles that are prevalent in other populations. This highlights the importance of using more than one pair of primers for amplification of each gene.

10The gene content of some KIR haplotypes and sequence of some KIR alleles strongly suggest that unequal crossing-over in the region accounts for a substantial amount of the diversity at this locus (2023). This could result in the production of unusual profiles where, for example, one or more of the framework genes are missing or chimeric genes are present.

11Some of the KIR loci exhibit strong negative and positive linkage disequilibrium between them. For example, some gene pairs such as KIR2DL2/2DS2, KIR2DL1/2DP1, and KIR3DL1/2DS4 are almost always found together across different haplotypes. This information is very useful in validation of the genotyping results.


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