Genome amplification of single sperm using
multiple displacement amplification
Zhengwen Jiang1, Xingqi Zhang2, Ranjan Deka1and Li Jin3,1,*
1Department of Environmental Health, Center for Genome Information, University of Cincinnati College of
Medicine, 3223 Eden Ave, Cincinnati, OH 45267, USA,2Department of Obstetrics and Gynecology,
Northwestern University Medical School, Chicago, IL, USA and3State Key Laboratory of Genetic
Engineering and Center for Anthropological Studies, School of Life Sciences and Morgan-Tan
International Center for Life Sciences, Fudan University, Shanghai, China
Received February 2, 2005; Revised April 11, 2005; Accepted May 17, 2005
Sperm typing is an effective way to study recombina-
tion rate on a fine scale in regions of interest. There
are two strategies for the amplification of single mei-
otic recombinants: repulsion-phase allele-specific
PCR and whole genome amplification (WGA). The
former can selectively amplify single recombinant
molecules from a batch of sperm but is not scalable
for high-throughput operation. Currently, primer
extension pre-amplification is the only method used
in WGA of single sperm, whereas it has limited capa-
city to produce high-coverage products enough for
the analysis of local recombination rate in multiple
large regions. Here, we applied for the first time a
recently developed WGA method, multiple displace-
ment amplification (MDA), to amplify single sperm
DNA, and demonstrated its great potential for produ-
reaction, 76 or 93% of loci can be amplified at least
2500- or 250-fold, respectively, from single sperm
DNA, and second-round MDA can further offer >200-
fold amplification. The MDA products are usable for a
variety of genetic applications, including sequencing
and microsatellite marker and single nucleotide poly-
morphism (SNP) analysis. The use of MDA in single
sperm amplification may open a new era for studies
on local recombination rates.
A detailed knowledge of linkage disequilibrium (LD)
patterns across the human genome was widely considered a
prerequisite for comprehensive association testing (1). Recent
data have shown that LD in human populations is highly
structured into discrete blocks with limited haplotype diversity
(2–5). This LD structure was believed to result from the inter-
play between recombination hotspots (3,5,6) and the demo-
graphic history of human populations (7,8). Little is known
about the role of recombination in shaping LD patterns in
populations, although statisticalapproaches may provide some
clues (9–11). The answer to this question may lie in compar-
crossovers. Sperm typing can identify the distribution of male
local meiotic recombination rate, which can at least partially
explain the LD pattern, as exemplified by Jeffreys et al. (3).
Two strategies have been taken for detecting highly local-
ized meiotic recombination hotspots in sperm. One is to
amplify single recombinant molecules using repulsion-phase
allele-specific PCR from a large batch of sperm DNA, fol-
lowed by localization of crossover sites (3,12–14). The other is
to scan thousands of single sperm cells to identify and localize
meiotic recombinants (15–17). The former is efficient for fine
mapping of crossover sites in a defined region of several kilo-
bases without laborious work on screening recombinants as
required in the latter. However, it can only be used in studies
on small regions of <10 kb and the workers have to be very
careful of possible contamination from artificial recombinants
resulting from template switching during PCR amplification
(18,19). For the second method, single sperm cells were first
pre-amplified by either multiple PCR or whole genome amp-
lification (WGA) to produce sufficient DNA for further mul-
tiple genotyping reactions to identify and localize meiotic
recombinants. Although multiple PCR has been more widely
used for single sperm analysis (20–22), WGA seems to be
more promising and preferred for fine mapping of meiotic
recombination sites because it can amplify many more marker
loci than multiple PCR. Primer extension pre-amplification
(PEP) was developed to amplify single sperm DNA on
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Shanghai 200433, China. Tel: +86 21 65642800; Fax: +86 21 55664388; Email: firstname.lastname@example.org
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Nucleic Acids Research, 2005, Vol. 33, No. 10e91
the whole genome level in 1992 (23) and first applied for
localization of recombination sites in 1994 (15). Recently,
Cullen et al. (16) used PEP to pre-amplify 20 031 single
sperm cells, followed by genotyping using 48 short tandem
repeat (STR) markers in a 3.3 Mb interval encompassing the
major histocompatibility complex (MHC). Unfortunately,
their study did not achieve high enough density to allow com-
parison with the LD map, owing to insufficient PEP products
and low marker density.
Recently, multiple displacement amplification (MDA),
using F29 DNA polymerase and random exonuclease-
resistant hexamer primers, has been demonstrated to be
very efficient for balanced amplification and generation of
long DNA products (>10 kb) from a small amount of DNA,
or directly from whole blood (24). The high processivity and
fidelity of F29 DNA polymerase provides an advantage to
MDA in terms of yield, accuracy and coverage over the other
PCR-based WGA methods (24,25). Amplification of single
lymphocytes or blastomeres using MDA has been successfully
carried out (26,27). Up to 92% coverage was estimated from
100 PCRs by detecting 20 different loci in five single lympho-
cytes, although at heterozygous loci, a high rate of allele
dropout (31%) was observed (26).
In this paper, we demonstrate that MDA can be utilized to
produce high-coverage and high-yield WGA products from
single sperm cells, and the resulting MDA products can be
used for a variety of genetic applications, including sequen-
cing and microsatellite marker (STR) and single nucleotide
polymorphism (SNP) analysis. It was estimated that 76% of
the genomic sequence can be amplified at least 2500-fold, or
93% at least 250-fold. We also showed that the second-round
MDA can further give an averaged 236-fold amplification.
MDA can therefore be quite useful for amplifying single
sperm DNA for a large amount of genotyping reactions and
this may open a new avenue for single sperm analysis.
MATERIALS AND METHODS
Sperm sample and genomic DNA preparation
Fertile men considering vasectomy were approached for
consent to enter the study. Once informed consent was
obtained, a blood sample and semen sample (by masturbation)
were collected. The blood sample (10 ml) was collected
into tubes with acid citrate dextrose (ACD) and centrifuged
at 3300 g at room temperature for 10 min. The intermediate
layer where white blood cells were concentrated was collected
and resuspended in phosphate-buffered saline (PBS) for
further processing for DNA analysis. Genomic DNA was
extracted from white blood cells using the standard phenol–
chloroform method. DNA concentration was determined using
a Hoefer DyNA Quant 200 Fluorometer.
Sperm cells were counted with a hemacytometer, diluted to a
concentration of either 0.8 or 3 cells/3 ml with PBS and 16
aliquots were prepared of each dilution. Three microliter of
diluted sperm cells were dispensed into 200 ml PCR tubes and
frozen at ?80?C overnight. An aliquot of 3.5 ml of freshly
prepared lysis solution (0.1 M DTT, 0.4 M KOH and 10 mM
EDTA) was then added, mixed well by gentle vortex and
incubated for 10 min on ice for eight aliquots of the dilution
of 3 cells/3 ml, or at 65?C for the other aliquots. Lysis was
stopped by adding 3.5 ml of neutralizing buffer (buffer B in
REPLI-g kit, Qiagen Inc.). The dilution of 3 cells/3 ml was
picked to test whether 65?C incubation could lyse sperm cells
better or not, and the dilution of 0.8 cells/3 ml was used to
obtain aliquots containing single sperm cells. Aliquots named
after S01, S02...S16 below were prepared from the dilution of
0.8 cells/3 ml
Multiple displacement amplification
WGA was achieved using REPLI-g? kit according to the
manufacturer’s manual (Qiagen Inc.). All samples were pre-
amplified by MDA. A PBS blank was included as a negative
control. A reaction in a total volume of 50 ml was performed
at 30?C overnight and then terminated at 65?C for 10 min.
Amplified DNA products were then stored at ?20?C. Dilu-
tions of 5- or 50-fold (referred as 1/5C0and 1/50C0, respect-
ively, below) were used for further sequencing, the coverage
test and microsatellite and SNP genotyping analysis. One
microliter of a 10-fold diluted S16 MDA product was used
as template for the second-round MDA.
PCR and sequencing analysis
In order to determine the aliquots that were successfully pre-
amplified by MDA, three genes—TOP1, P53 and CYP1A2—
were selected for PCR testing using 1 ml of 1/5C0MDA prod-
uct. Primers used are listed in set A of Table 1.A 20 ml mixture
was prepared for each reaction and included 1· HotStarTaq
buffer, 2.5 mM Mg2+, 0.2 mM dNTP, 0.3 mM of each primer,
1 U HotStarTaq polymerase (Qiagen Inc.) and 1 ml template
DNA. The cycling program was 95?C for 15 min; 40 cycles of
94?C for 15 s, 56?C for 30 s, 72?C for 1 min; 72?C for 2 min.
Amplified fragments representative of the three genes (TOP1,
P53 and CYP1A2) were 1080, 643 and 550 bp in length,
respectively. PCR products were checked on 1.5% agarose
gels. For the aliquots of the 0.8 cells/3 ml dilution, those
MDA products in which at least one of the three genes got
amplified were selected for further analyses.
A total of 12 genes, including TOP1, P53, CYP1A1,
PIK3CA, C6orf195, DKKL1, SHH, ADCYAP1, MSH2,
PTEN, PMS2 and CAT, were examined to estimate amplifi-
cation coverage. Almost all genes are located on different
chromosomes, except for SHH and PMS2, which are both
on chromosome 7 (PMS2 on the p arm and SHH on the q
arm). The standard PCR method described above was used to
amply 12 sequences of length 162–351 bp, one for each gene,
from 1 ml of 1/50C0MDA product. Primers used to amplify
these fragments are listed in set B of Table 1. Five microliter
PCR products were then loaded on 1.5% agarose gels and
checked for the presence of target fragments. For those sam-
ples in which some primers did not work, the 1/5C0MDA
products were submitted to PCR again with the failed primers.
To assess the fidelity of WGA from single cells, we used
another four pairs of primers (set C in Table 1) to amplify
four DNA fragments (two in TOP1 and two in CYP1A1) with
a total length of ?3.3 kb, and these fragments were then
sequenced using ABI Big Dye Terminator V3 kit and
ABI3100 capillary sequencer (AppliedBiosystems, Foster
e91Nucleic Acids Research, 2005, Vol. 33, No. 10
PAGE 2 OF 9
Lam,A.C., Guja,C., Ionescu-Tirgoviste,C., Undlien,D.E. et al. (2004)
Comparative high-resolution analysis of linkage disequilibrium and tag
single nucleotide polymorphisms between populations in the vitamin D
receptor gene. Hum. Mol. Genet., 13, 1633–1639.
6. Kauppi,L.,Sajantila,A.and Jeffreys,A.J. (2003)Recombination hotspots
rather than population history dominate linkage disequilibrium in the
MHC class II region. Hum. Mol. Genet., 12, 33–40.
7. Wang,N., Josh,A.M., Zhang,K., Chakraborty,R. and Jin,L. (2002)
Distribution of recombination crossovers and the origin of haplotype
blocks: the interplay of population history, recombination, and
mutation. Am. J. Hum. Genet., 71, 1227–1234.
8. Zhang,K., Akey,J.M., Wang,N., Xiong,M., Chakraborty,R. and Jin,L.
of linkage disequilibrium: an act of genetic drift. Hum. Genet.,
9. Wiehe,T., Mountain,J., Parham,P. and Slatkin,M. (2000) Distinguishing
recombination and intragenic gene conversion by linkage disequilibrium
patterns. Genet. Res., 75, 61–73.
10. Reich,D.E., Schaffner,S.F., Daly,M.J., McVean,G., Mullikin,J.C.,
Higgins,J.M., Richter,D.J., Lander,E.S. and Altshuler,D. (2002) Human
genome sequence variation and the influence of gene history, mutation
and recombination. Nature Genet., 32, 135–142.
11. Phillips,M.S., Lawrence,R., Sachidanandam,R., Morris,A.P.,
Balding,D.J., Donaldson,M.A., Studebaker,J.F., Ankener,W.M.,
Alfisi,S.V., Kuo,F.S. et al. (2003) Chromosome-wide distribution of
haplotype blocks and the role of recombination hot spots. Nature Genet.,
12. Jeffreys,A.J., Murray,J. and Neumann,R. (1998) High-resolution
recombination hotspot. Mol. Cell., 2, 267–273.
of haplotype diversity and meiotic crossover in human TAP2
recombination hotspot. Hum. Mol. Genet., 9, 725–733.
14. May,C.A., Shone,A.C., Kalaydjieva,L., Sajantila,A. and Jeffreys,A.J.
(2002) Crossover clustering and rapid decay of linkage disequilibrium in
the Xp/Yp pseudoautosomal gene SHOX. Nature Genet., 31,
15. Hubert,R., MacDonald,M., Gusella,J. and Arnheim,N. (1994) High
resolution localization of recombination hot spots using sperm typing.
Nature Genet., 7, 420–424.
16. Cullen,M., Perfetto,S.P., Klitz,W., Nelson,G. and Carrington,M. (2002)
High-resolution patterns of meiotic recombination across the
human major histocompatibility complex. Am. J. Hum. Genet., 71,
Direct measurement of the male recombination fraction in the human
beta-globin hot spot. Hum. Mol. Genet., 11, 207–215.
18. Pa ¨a ¨bo,S., Irwin,D.M. and Wilson,A.C. (1990) DNA damage promotes
jumping between templates during enzymatic amplification. J. Biol.
Chem., 265, 4718–4721.
19. Zangenberg,G., Huang,M.M., Arnheim,N. and Erlich,H. (1995) New
HLA-DPB1alleles generatedby interallelicgeneconversiondetectedby
analysis of sperm. Nature Genet., 10, 407–414.
20. Furlong,R.A., Goudie,D.R., Carter,N.P., Lyall,J.E., Affara,N.A. and
Ferguson-Smith,M.A. (1993) Analysis of four microsatellite markers on
the long arm of chromosome 9 by meiotic recombination in flow-sorted
single sperm. Am. J. Hum. Genet., 52, 1191–1199.
21. Girardet,A., Lien,S., Leeflang,E.P., Beaufrere,L., Tuffery,S., Munier,F.,
Arnheim,N., Claustres,M. and Pellestor,F. (1999) Direct estimation of
the recombination frequency between the RB1 gene and two closely
linked microsatellites using sperm typing. Eur. J. Hum. Genet., 7,
22. Park,C., Frank,M.T. and Lewin,H.A.(1999) Fine-mapping of a region of
using sperm typing and meiotic breakpoint analysis. Genomics, 59,
23. Zhang,L., Cui,X., Schmitt,K., Hubert,R., Navidi,W. and Arnheim,N.
(1992) Whole genome amplification from a single cell: implications for
genetic analysis. Proc. Natl Acad. Sci. USA, 89, 5847–5851.
24. Dean,F.B., Hosono,S., Fang,L., Wu,X., Faruqi,A.F., Bray-Ward,P.,
Sun,Z., Zong,Q., Du,Y., Du,J. et al. (2002) Comprehensive human
genome amplification using multiple displacement amplification.
Proc. Natl Acad. Sci. USA, 99, 5261–5266.
25. Lasken,R.S. and Egholm,M. (2003) Whole genome amplification:
abundant supplies of DNA from precious samples or clinical specimens.
Trends Biotechnol., 21, 531–535.
26. Handyside,A.H., Robinson,M.D., Simpson,R.J., Omar,M.B.,
Shaw,M.A., Grudzinskas,J.G. and Rutherford,A. (2004) Isothermal
whole genome amplification from single and small numbers of cells: a
new era for preimplantation genetic diagnosis of inherited disease.
Mol. Hum. Reprod., 10, 767–772.
and Ozand,P. (2004) Multiple displacement amplification on single cell
and possible PGD applications. Mol. Hum. Reprod., 10, 847–852.
28. Carter,K.L., Robertson,B.C. and Kempenaers,B. (2000) A differential
eggs. Mol. Ecol., 9, 2149–2150.
29. Cheung,V.G. and Nelson,S.F. (1996) Whole genome amplification
using a degenerate oligonucleotide primer allows hundreds of genotypes
to be performed on less than one nanogram of genomic DNA.
Proc. Natl Acad. Sci. USA, 93, 14676–14679.
30. Klein,C.A., Schmidt-Kittler,O., Schardt,J.A., Pantel,K., Speicher,M.R.
and Riethmuller,G. (1999) Comparative genomic hybridization, loss of
heterozygosity, and DNA sequence analysis of single cells. Proc. Natl
Acad. Sci. USA, 96, 4494–4499.
31. Wells,D., Sherlock,J.K., Handyside,A.H. and Delhanty,J.D. (1999)
Detailed chromosomal and molecular genetic analysis of single cells by
whole genome amplification and comparative genomic hybridisation.
Nucleic Acids Res., 27, 1214–1218.
LM-PCR permits highly representative whole genome amplification
tissue sections. Diagn. Mol. Pathol., 13, 105–115.
33. Stoecklein,N.H., Erbersdobler,A., Schmidt-Kittler,O., Diebold,J.,
Schardt,J.A., Izbicki,J.R. and Klein,C.A. (2002) SCOMP is superior
to degenerated oligonucleotide primed-polymerase chain reaction for
global amplification of minute amounts of DNA from
microdissected archival tissue samples. Am. J. Pathol., 161, 43–51.
Whole genome amplification of DNA from laser capture-microdissected
tissue for high-throughput single nucleotide polymorphism and short
tandem repeat genotyping. Am. J. Pathol., 164, 23–33.
35. Wang,G., Brennan,C., Rook,M., Wolfe,J.L., Leo,C., Chin,L., Pan,H.,
Liu,W.H., Price,B. and Makrigiorgos,G.M. (2004) Balanced-PCR
amplification allows unbiased identification of genomic copy changes in
minute cell and tissue samples. Nucleic Acids Res., 32, e76.
36. Wang,G., Maher,E., Brennan,C., Chin,L., Leo,C., Kaur,M., Zhu,P.,
Rook,M., Wolfe,J.L. and Makrigiorgos,G.M. (2004) DNA amplification
method tolerant to sample degradation. Genome Res., 14, 2357–2366.
37. Canceill,D., Viguera,E. and Ehrlich,S.D. (1999) Replication slippage of
different DNA polymerases is inversely related to their strand
displacement efficiency. J. Biol. Chem., 274, 27481–27490.
38. Viguera,E., Canceill,D. and Ehrlich,S.D. (2001) In vitro replication
slippage by DNA polymerases from thermophilic organisms.
J. Mol. Biol., 312, 323–333.
39. Blanco,L., Bernad,A., Lazaro,J.M., Martin,G., Garmendia,C. and
polymerase. Symmetrical mode of DNA replication. J. Biol. Chem., 264,
PAGE 9 OF 9
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