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We have identified tens of thousands of short
extrachromosomal circular DNAs (microDNA) in mouse
tissues as well as mouse and human cell lines. These
microDNAs are 200-400 bp long, derived from unique
non-repetitive sequence and are enriched in the 5'
untranslated regions of genes, exons and CpG islands.
Chromosomal loci that are enriched sources of microDNA
in adult brain are somatically mosaic for microdeletions
that appear to arise from the excision of microDNAs.
Germline microdeletions identified by the "Thousand
Genomes" project may also arise from the excision of
microDNAs in the germline lineage. We have thus
identified a new DNA entity in mammalian cells and
provide evidence that their generation leaves behind
deletions in different genomic loci.
Single nucleotide polymorphisms and copy number variations
are known sources of genetic variation between individuals
(1–5), but there is also great interest in variations that arise
during generation of somatic tissues like the mammalian
brain, leading to genetic mosaicism between somatic cells. To
identify sites of intramolecular homologous recombination
during brain development, we searched for extrachromosomal
circular DNA (eccDNA) derived from excised chromosomal
regions in normal mouse embryonic brains.
We purified eccDNA from nuclei of embryonic day 13.5
(ED13.5) mouse brain, and removed linear DNA by digestion
with an ATP-dependent exonuclease (6) (fig. S1, table S1 and
SOM Methods). Multiple displacement amplification (MDA)
with random primers (7, 8) enriched circular DNA by rolling
circle amplification. The linear products of MDA were
sheared to 500 bp fragments, cloned into a plasmid and
clones sequenced. Out of 93 clones, 73 contained direct
repeats of several hundred base-pairs (fig. S2), as would be
expected from rolling circle amplification of circles that are a
few hundred bp long. Only one copy of the repeat sequence
was present in the mouse genome (figs. S2, S3), indicating
that the direct repeats were derived from unique non-
repetitive DNA in the genome and could have been generated
by rolling circle amplification of a circularized form of
Three sequences that appeared >2 times in the 73 clones
were chosen to confirm the circular nature of the
extrachromosomal DNA before any MDA. Outward-directed
primers yielded PCR products from 10% of total
extrachromosomal DNA (without any MDA), but not from
linear genomic DNA for two out of the three sequences (Fig.
1a). The PCR products from outward-directed primers had
the same junctions as seen between repeats in the MDA
products of the extrachromosomal DNA (Fig. 1b). These
results are consistent with the circularization of linear
genomic DNA to produce extrachromosomal circular DNA.
To determine the number, size, nature and source of these
short eccDNA, we isolated eccDNA from ED13.5 mouse
brain, heart and liver, adult mouse brain, mouse (NIH3T3),
and human (HelaS3 and U937) cell lines (table S1).
Following MDA of the eccDNA, ~500 bp fragments of the
amplified DNA were subjected to paired-end sequencing. As
a negative control, chromosomal DNA from embryo mouse
brain nuclei was treated in an identical manner to the
eccDNA fraction. We also examined eccDNA fraction from
S.cerevisiae by exactly the same procedure (SOM text).
Circular DNAs were identified by two different algorithms
that were dependent on the identification of junctional tags
created by the circularization (fig. S4 and SOM Methods).
Tens of thousands of unique sequences in the genome were
identified as yielding extrachromosomal circular DNA (table
S2) and their total yield was 0.1- 0.2% weight of
chromosomal DNA in normal tissue. In contrast, the negative
control mouse chromosomal DNA yielded only 114 circles,
all arising from contamination by extrachromosomal DNA,
because the same circles were abundant in the ecc libraries.
No circles were detected in the S. cerevisiae
The circular DNA from mouse tissues and cell lines were
80-2000 bp long, though >50% were in the 200-400 bp range
with clear peaks in the brain and liver at ~200 and ~400 bp
Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues
Yoshiyuki Shibata,1* Pankaj Kumar,1* Ryan Layer,1 Smaranda Willcox,2 Jeffrey R. Gagan,1 Jack D. Griffith,2
1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia,
USA. 2Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: email@example.com
on March 19, 2012
(Fig. 1c). In the two human cancer cell lines, where we
identified many more circular DNAs, the length distribution
also peaked at 200 and 400 bp but had additional peaks with a
periodicity of 150 bp (Fig. 1c). The circular DNAs were
uniquely mapped to the genome and were not derived from
repetitive sequences. These DNAs were therefore different
from previously reported eccDNAs that were a few hundred
to millions of bases long and derived from chromosomal
repetitive sequences, intermediates of mobile elements or
viral genomes (9, 10). Based on their small size and
derivation from unique genomic sequence we named this
family of DNA as microDNA.
To detect the 200-400 base long microDNAs in cells by a
fourth method, the eccDNA fraction from mouse brain, after
exonuclease digestion but without rolling circle amplification,
was directly examined by electron microscopy. Double-
stranded microDNA that are several hundred bp long were
easily detected (Fig. 1d, fig. S5a, b). We also found single-
stranded microDNA visualized after the treatment of DNA by
single-stranded DNA binding protein, gp32 (Fig. 1e, fig. S5a,
b). The double- and single-stranded microDNAs were
equivalent in number. More than 98% of the circular DNA
from mouse brain was small (<1 kb) (SOM text), making this
the dominant population of eccDNA in normal somatic tissue.
Thus PCR with outward directed primers (Fig. 1a, b) or
electron microscopy (Fig. 1d, e) on extrachromosomal DNA
fraction without MDA confirmed the presence of short circles
that were revealed by Sanger sequencing (figs. S2, S3) or
ultrahighthroughput sequencing (Fig. 1c, fig. S4) of MDA
The sources of the microDNAs from the embryo mouse
brain (EMB1) were highly enriched in genic regions,
especially 5’ regions of genes, exons, and CpG islands (Fig.
2a). A similar trend was also observed in microDNA from
other mouse tissues and mouse and human cell lines (fig. S6).
Furthermore, the 55% GC content of microDNAs is higher
than the 50% GC content of the immediate upstream or
downstream flanking regions and the 45% GC composition of
the entire genome (Fig. 2b, figs. S7 and S8). The starts and
ends of the circles revealed 2-15 bp direct repeats of micro-
homology (Fig. 2c, fig. S9). In the EMB1 library 37% of the
microDNA has this micro-homology, while in the random
model (SOM Method) <3% of the shuffled microDNAs had
micro-homology of ≥2 bp near the ends (p<0.0001) (Fig. 2d).
Direct repeats were similarly present at the ends of the
microDNA from all mouse tissues and human cell-lines (Fig.
The lengths of microDNAs from cancer cell lines show a
pronounced periodicity of 150 bp, (Fig. 1c) consistent with
the possibility that nucleosome wrapping of DNA may
contribute to microDNA generation. In addition, though
microDNAs are rich in GC content, AA/AT/TT dinucleotides
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were found along the length of many circles with a
periodicity of 9-11 bp (example in Fig. 2e). GC richness
periodically punctuated by AA/AT/TT dinucleotides is a
feature of sequences preferentially assembled into
nucleosomes (11, 12). Around 50-60% of microDNAs in the
different libraries overlapped by ≥15 bases with 25-mer tags
marking the locations of positioned nucleosomes determined
in the mouse liver (13) (Fig. 2e and fig. S10) (p< 0.001 in “t”
test from random distribution).
The features of these microDNAs are completely different
from the sequences obtained from chromosomal DNA,
suggesting that the specific characteristics of microDNA are
not an artifact of random sampling of cellular DNA by
highthroughput sequencing (fig. S11a-c and SOM Text).
Cells that release a double-stranded circular DNA may be
expected to suffer a microdeletion in the source genomic
locus. A search for such microdeletions is complicated by the
fact that different cells are likely to yield different
microDNAs, so that a tissue will be mosaic for
microdeletions. We therefore selected two genomic loci that
yielded microDNAs in multiple brain libraries. One was 20
kb at the 5' end of the KCNK3 gene in chromosome 5
(30,890,697-30,910,805, NCBI37/mm9) enriched by PCR
(Fig. 4b), and another was 160 kb on chromosome 10
(80,213,587-80,372,454, NCBI37/mm9) enriched by
Anchored ChromPET (14). The strategy for finding
microdeletions in the selected loci is given in Fig. 3a and the
SOM Methods. A total of thirty deletions were detected (23
from the KCNK3 locus and 7 from the chromosome 10 locus)
(Fig. 3a and fig. S13). Direct repeats were observed at both
ends of 25 of the 30 microdeletions (Fig. 3b and fig. S13).
The GC composition, length distribution and AA/AT/TT
periodicity of the microdeletions were also similar to that
observed for the microDNA (Fig. 3c, figs. S12 and S13). The
results suggest that microdeletions occur in an average of 1 in
2000 chromosomal DNA molecules (SOM text) at
susceptible genomic loci in somatic tissues, giving rise to
genetic variability between individual normal somatic cells.
The widespread occurrence of microDNAs led us to
wonder whether microdeletions in germ line sequence could
also result from the excision of microDNAs. In fact the
germline deletions of <1000 bp reported in the Thousand
Genomes project (15) had features similar to that of
microDNAs (Fig. 4a-d and SOM Text). Briefly, the germline
microdeletions peaked in length at 100 and 350 bp, were
enriched in exons, 5'UTRs and CpG islands, were rich in GC
content and had a high frequency of short direct repeats
flanking the deleted fragments. This close overlap between
the nature of the sequences lost in germline microdeletions
and the microDNAs reported in this paper suggest that these
deletions are also generated by the excision and loss of
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Unlike formerly described eccDNA (9, 10), microDNAs
are small, map to unique DNA sequence and appear from
genes. Very short direct repeats at the starts and ends of
microDNAs suggest that fork stalling/template switching
during replication/repair or microhomology-mediated repair
may produce microDNAs. Circularization of microDNAs
could be facilitated by the wrapping of DNA around
positioned nucleosomes. The known correspondence of
positioned nucleosomes with 5' ends of genes could explain
the enrichment of microDNAs from the 5' ends of genes.
MicroDNAs could also originate as displaced Okazaki
fragments from replication forks collapsed at strongly bound
nucleosomes or GC-rich DNA. Single-stranded microDNAs
may arise from such ligated Okazaki fragments, from deletion
of excess DNA produced by replication slippage or from
nuclease digestion of nicked double-stranded circles.
However, the microdeletions detected in genomic loci most
likely arise from excision of double-stranded circles. The
generation of microDNAs and microdeletions may produce a
large pool of individual-specific or somatic-clone-specific
copy-number variations of small segments of the genome.
The genetic mosaicism in somatic tissues may lead to
functional differences between cells in a tissue. Finally
persistent microDNAs may provide the extrachromosomal
genetic "cache" that has been postulated to account for non-
Mendelian genetics in plants (18).
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Acknowledgments: 1.This work was supported by R01
CA60499 and GM84465 to AD, and GM31819 and
ESO13773 to JDG. We thank all members of the Dutta
Lab for helpful discussions, and A. Prorock for assistance
with DNA sequencing. Accession numbers for the
sequence data: GSE36088 (Extrachromosomal
microDNAs and chromosomal microdeletions in normal
tissues); GSM880919 (Embryo mouse brain sample 1);
GSM880920 (Embryo mouse brain sample 2);
GSM880921 (Embryo mouse brain sample 3);
GSM880922 (Embryo mouse heart); GSM880923
(Embryo mouse liver); GSM880924 (Adult mouse brain);
GSM880925 (NIH3T3 mouse cell lines); GSM880926
(Embryo mouse brain sample); GSM880927 ( HeLaS3
human cell lines); GSM880928 (U937 human cell lines).
Supporting Online Material
Materials and Methods
Fig. S1 to S13
Table S1 and S2
29 August 2011; accepted 24 February 2012
Published online 8 March 2012; 10.1126/science.1213307
Fig. 1. Tiny circular DNA are detected in the
extrachromosomal DNA fraction. a. Outward-directed PCR
primers (Out) amplified DNA fragments from
extrachromosomal DNA (E), but not from genomic DNA (G).
DNA was amplified by inward-directed PCR primers (In)
from both (E) and (G). b. Sequencing of fragments amplified
by Out primers on extrachromosomal fraction. Underlined
sequences indicate primers. Junctions between red and blue
sequences were the same as that observed in clones in fig. S2.
c. Length distribution of microDNAs from various tissues and
cell lines. The library abbreviations are explained in SOM. d.
EM of double-stranded microDNA examined by the
cytochrome c drop spreading method (16) (50 nm = 150 bp).
e. EM of single-stranded microDNA after binding with the T4
gene 32 single stranded DNA binding protein (17).
Fig. 2. Properties of the loci that give rise to microDNAs. a.
Enrichment of microDNAs observed in the indicated genomic
region relative to the expected percentage based on random
distribution. b. Distribution of GC composition in
microDNAs in the EMB1 library and their up- and down-
stream regions (of same length as microDNA). Vertical line:
the genomic average GC content. c. Presence of micro
homology near the start and end of a microDNA.
"MicroDNA island (blue curve)" is a contiguous stretch of
the genome to which the PE-tags map uniquely and correctly.
Direct repeats of 2-15 bp (red letters) were observed at the
junction of the circle (Upper case) with flanking genomic
DNA (Lower case). d. Direct repeats are enriched in different
microDNA libraries compared to the random model (RM),
generated from the EMB1 sequences. e. Intersection of
microDNAs from EMB1 with positioned nucleosome-
occupied regions in the mouse liver (13). Obs: observed
overlap with nucleosome-occupied DNA. Exp: expected
overlap of 1000 randomizations of each microDNA in the
library (p<0.0001). A similar enrichment is seen with other
microDNA libraries (fig. S10).
Fig. 3. Microdeletions in genomic loci known to yield
microDNAs. a. Algorithm for finding microdeletions in
genomic DNA. Details in SOM. b. Micro-deletions found in
the KCNK3 locus. DNA spanning the indicated locus was
amplified from 200,000 copies of 6 month old mouse brain
genomic DNA, and paired-end-sequenced. White square is
KCNK3 exon1 and solid line is KCNK3 intron1. Blue
squares are positions of microDNAs identified in three
independent embryonic brain libraries, and red squares are
microdeletions found in the genome in this study. c. Direct
repeats observed near the junctions of microdeletions. d. GC
composition of the microdeletions identified in the two loci.
The deleted sequences were rich in GC content compared to
the genomic average of 46%.
Fig. 4. Germline deletions of <1000 bp in the Thousand
Genomes Project have properties similar to microDNAs. a.
Length distribution peaks at 100 bp and 350 bp. b. Deletions
in genic areas are enriched in 5'UTRs, exons, CpG islands
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and regions 200bp upstream from genes. c. GC content of
deletion and up-stream and down-stream regions is greater
than genomic average. The up-stream and down-stream
sequence was of same length as the deletions. d. 70% of the
microdeletions had flanking direct repeats. Length
distribution of the direct repeats is shown. Direct repeats ≥15
bp are shown at 15 bp.
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