1. High-Resolution Mapping of Crossovers in Human Sperm Defines a Minisatellite- Associated Recombination Hotspot 
Alec J Jeffreys, John Murray, Rita Neumann 
Molecular Cell 
Volume 2, Issue 2, Pages (August 1998)
DOI: /S (00)

2. Figure 1
Haplotype Diversity in DNA Flanking the Unstable End of Human Minisatellite MS32
(A) Location of base substitutional polymorphisms relative to the MS32 repeat array. This region also contains three Alu elements (white boxes) inserted into a diverged L1 element (light shading) plus a diverged RTLV-1 retroviral LTR sequence from which MS32 has expanded (dark box) (Armour et al. 1989; Gray and Jeffreys 1991).
(B) Haplotypes identified in 37 Caucasian semen donors. Closed circles correspond to alleles AlA, BaG, RsT, BcT, XbT, AwC, BsG, RtA, M1T, SaC, H1G, HfC, and H2C, and open circles to each alternative allele. There was no significant association between MS32 repeat array length and flanking haplotype (ANOVA, F2,71 = 0.57; P = 0.567); for example, the 30 identical haplotypes in group a are linked to MS32 alleles ranging from 48 to 900 repeats in length and covering most of the range of allele lengths seen in Caucasian populations (data not shown). Haplogroups a–c are in Hardy-Weinberg equilibrium (not shown).
(C) Patterns of linkage disequilibrium determined from these 74 haplotypes. The standardized disequilibrium coefficient (Hill and Robertson 1968) was determined for each adjacent pair of markers, either omitting marker Rs (thick line) or including it (thin line).
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 Detecting Crossover Events near MS32
(A) Haplotypes of MS32 alleles in semen donor, showing positions, not to scale, of flanking polymorphisms and PCR primers. Sperm molecules similar or identical in length to the smaller allele but with distal upstream markers from the larger allele will include (i) recombinants in the flanking DNA, (ii) equal crossovers in the repeat array, (iii) interallelic unequal crossovers, and (iv) large deletions from the larger allele.
(B) Detecting recombinants. Sperm DNA was digested with PstI, which cleaves 5385 bp upstream and 474 bp downstream of MS32, and the smaller allele 2 (haplogroup c) recovered by gel electrophoresis. This allele was mixed with sperm DNA from a control individual heterozygous for 220- and 34-repeat haplogroup c alleles (X) and preamplified in batches of 2100 molecules per allele with universal primers −3.5F and 32D1. Primary PCR products were reamplified with allele-specific primer BaG plus universal primer 32A, and the resulting secondary PCR products further reamplified with allele-specific primer BcT and universal primer 32E. PCR products were detected by Southern blot hybridization following agarose gel electrophoresis. The tertiary PCRs show examples of isometric recombinants derived from allele 2 plus new length mutants and one example of a molecule of the larger allele still present in the fractionated DNA (arrowed).
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3 Locating Crossover Breakpoints near MS32 by RFLP Analysis
5′-flanking DNA from tertiary PCRs 1–10 containing recombinants was reamplified using the allele-specific primer XbT plus the universal primer 32PR (Figure 2) and analyzed for markers Bs–H2. L, larger allele; S, smaller allele.
Interpretation: PCRs 1, 6, and 10 contain flanking markers exclusively from the larger allele. Numbers 4 and 9 contain Bs–M1 markers from the larger allele and Sa–H2 from the smaller, locating crossover points to the M1–Sa interval. Similarly, number 7 has recombined between Hf and H2, and number 8 between Sa and Hf. Number 2 is mixed at the Hf site and therefore contains two crossovers, one between Sa and Hf, and one between Hf and H2. Numbers 3 and 5 are similarly mixed, at the Sa and H2 sites, respectively.
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4 Meiotic Crossover Activity in and near Minisatellite MS32
Alleles 1–4 were purified from sperm DNA from four individuals and assayed for recombination events upstream of the repeat array (dark shading), plus equal crossovers (medium shading) and unequal crossovers (no shading) within the array. These data were determined from surveys of 180,000, 200,000, 260,000, and 570,000 molecules of alleles 1–4, respectively. The crossover frequency in each interval between adjacent heterozygous markers is expressed as the ratio of the number of recombinants observed (after Poisson correction; numbers indicated above the histogram) to that expected from the genome average male recombination frequency of 8.9 × 10−3 per Mb (dotted line). Crossovers within the repeat array are binned into 6-repeat (174 bp) intervals. For unequal crossovers, the point of recombination is taken as the mean of the breakpoint locations in the two recombining alleles. The mean rates of unequal and equal crossover within the repeat array (6.3 × 10−5 and 3.1 × 10−5 per molecule, respectively) were similar to those established previously for other men using more proximal allele-specific primers (Jeffreys et al. 1998). Unequal crossover frequencies will be somewhat underestimated due to the loss on size fractionation of mutants showing a large change in repeat copy number; such mutants are, however, rare (Jeffreys et al. 1998).
Haplotypes: the flanking haplotypes at Al, Ba, Rs, Bc, Xb, Aw, Bs, Rt, M1, Sa, H1, Hf, H2, and O1 are given in order for each allele and its partner in each semen donor, together with allele length and four-state MVR code (repeat types e, E, y, Y, o; only the first and last 15 repeats shown for brevity). Allele 1, T-T-G-C-C-T-A-G-C-T-G-T-C-G, 40 repeats, eEEEEEEeEEEEEEe…yyeeeyeyyeyeEYY; partner, A-G-T-T-T-C-G-A-T-C-G-C-C-G, 420 repeats, YeEeEeEeEeEeEeE… EYYYYEYEEEYYYYY. Allele 2, T-T-G-C-C-T-A-G-C-T-G-T-T-G, 73 repeats, EeEEEeEEEyEEYEy…eeeeeeeeeeeeeYY; partner, A-G-T-T-T-C-G-A-T-C-G-C-C-G, 300 repeats, eeeeYeeYyeeEYEE… YYYYEYEoEYYYYYY. Allele 3, T-T-G-C-C-T-A-G-C-T-G-T-T-G, 144 repeats, EEeeeeYeEeEEEEe…eeeeeeeeeeeeeYY; partner, A-G-T-T-T-C-G-A-T-C-G-C-C-G, 450 repeats, YYeYYyeeeeeeeEe… EEE?YEEYEEYYYYY. Allele 4, T-T-T-C-C-T-A-A-C-C-G-T-T-C, 38 repeats, eEEYYEyYYoEEoEE…yyYYYEYEYYYYYYY; partner, A-G-T-T-T-C-G-A-T-C-G-C-T-G, 770 repeats, EYEEeeeeEeeeeeo… YYEYYYYEYEYYYYY.
Molecular Cell 1998 2, DOI: ( /S (00) )