Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

Abstract

Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.

Figure 1.

Schematic illustration of RQPS. A RQPS probe, which consists of a DNA fragment from the query gene physically linked to a DNA fragment from a reference gene, both containing artificially designed SNVs, is mixed with genomic DNA samples to attain a molar ratio between 1:9 and 9:1. The molar ratio of R-SNV, Q-SNV between probe and sample genomic DNA in each mixture is accurately determined by quantitative pyrosequencing. If the query gene is diploid, %G/%A = T%/C% (wild type, WT in the diagram). If one copy of the query gene is lost, %G/%A < T%/C% (loss of homozygosity, LOH in the diagram). If multiple copies of the query gene are present, then %G/%A > T%/C%. See text for details.

Figure 2.

RQPS accurately determines gene copy number. (A) Illustration of the Notch2-Pgk1 probe with SNVs introduced to differentiate it from the genomic counterparts. In the RQPS probe, the reference Notch2 fragment has a G-to-C SNV while Pgk1 has a C-to-G SNV. The fragment containing the Notch2 SNV was amplified with a three-primer system: (1) the forward tailed primer F1 with a complementary region to (2) the universal biotinylated primer (UBP), and (3) the untailed reverse primer R1. The fragment containing the Pgk1 SNV was amplified with the same UBP primer and two gene-specific primers (untailed forward primer F2 and a tailed reverse primer R2). The biotinylated PCR product was purified with streptavidin-coated Sepharose beads and sequenced with a sequence primer (S1 for Notch2 and S2 for Pgk1, respectively) hybridizing near the desired SNV. (B) Example of a readout (pyrogram) from a Biotage PSQ96-MA machine. Two probe/genomic DNA mixtures were prepared in this example. Reference and query SNVs were quantified by pyrosequencing of each mixture; the relative ratio is shown. Note that a G, instead of a C, determines the ratio of the RQPS probe in each mixture because the sequencing primer for the Notch2 reference gene binds to the minus strand. (C) The plot of m/(1 − m) against n/(1 − n) gives a line that passes (0, 0) with slope k = 0.59, which suggests that there is one copy of Pgk1 in this mouse and hence it is a male. (D) RQPS accurately assigns gender by determining the copy number of the X-linked Pgk1 gene. All male mice show a k-value of ∼0.6 and all females ∼1.2. (E) Statistical analysis of k-values from 18 animals tested demonstrates the robustness of RQPS. (F) RQPS accurately measures the copy number of exon 30 from the Notch1 gene, encoding a part of the Notch1 intracellular domain, in five different lines of mice that are known to have (a) one, (b) two, (c,e) three, and (d) four copies of the NICD1, respectively. When the N1 + /▵1;Gt(ROSA)26Sor^Notch/Notch mouse is used, owing to a deletion removing exon 30 (query) and exon 23 (reference) from one allele (Conlon et al. 1995), three copies of N1 ICD produced k = 3.1 since a haploid reference gene was used (e).

Figure 3.

RQPS differentiates homozygous from heterozygous transgenic animals. (A) RQPS on four Pax3-Cre animals. In this experiment, four different dilutions of RQPS probe were made with genomic DNA samples from each mouse; this was done to demonstrate that any two concentrations would have worked as well. Note the very small standard deviations for each data point, indicating that triplicates or duplicates are not necessary. Pyrosequencing of each mixture was performed in triplicate, and the average values were plotted; the standard deviation of each data point is shown. (B) RQPS of Tet-O-Cre mice. The copy number of the Tet-O-Cre transgene was measured in four animals. Four different mixtures were pyrosequenced in duplicate for each sample. RQPS detected two homozygotes and two heterozygotes, with about two and four copies of the transgene, respectively.

Figure 4.

Identifying HR clones in targeted ES cells using RQPS. (A) Refined screening of 32 ES cells falling within group A (Fig. 5B). Two different dilutions of RQPS probe were made for each ES cell genomic DNA sample and pyrosequenced. The black bar represents HR candidates (clone ID shown) with k-values indicating loss of one Notch1 allele. R² values for a zero intercept line are also plotted. (B) All HR candidates identified in A were confirmed by Southern blot, each containing the WT allele fragment (6 kb) and the targeted allele fragment (4.6 kb) expected after KpnI digestion.

Figure 5.

Pyroscreening of artificially introduced SNVs in targeting constructs dramatically enriched for positive HR clones. (A) Schematic illustration of pyroscreening. Artificially designed SNVs are introduced into targeting constructs: one in the HA (∼50 bp away from payload) and another one in the mouse Pgk1 promoter driving the expression of the selection marker. Different fates of the targeting construct in ES cells (HR, homologous recombination; RI, random integration) yield different ratios of the two linked SNVs to their endogenous counterparts. See text for details. (B) Pyroscreening of ES cells electroporated with (black dots) N1∷CreERT2, (green squares) N2∷cre, and (blue triangles) N1–N2. ES cells cluster into different groups. (Group A) Both HA and PGK SNV ratios fall between 40% and 60%, which suggests these are possible HR candidates. (Group B) HA SNV ratio ∼33% and PGK ∼40% suggest one copy of random integration. (Group C) The ratio suggests that these clones retained only one copy of the neomycin selection gene. (Group D) Poorly growing ES cells with heavy, G418-resistant MEF contamination. (Group E) ES cells that may have experienced multiple integrations. Note that nine out of 10 positive clones from (red dots) N1∷CreERT2, one out of one from (red square) N2∷Cre, and four of four from (red triangles) N1-N2 fall within group A. (C) When only one SNV is used for pyroscreening, the enrichment for positive clones is roughly threefold. This is increased to more than fourfold when two SNVs are combined.