Additional file 1: of Differential gene expression profiling of endometrium during the mid-luteal phase of the estrous cycle between a repeat breeder (RB) and non-RB cows

Table S1. List of up- and down-regulated genes in CAR of ipsilateral uterine horns of RB cows (n = 4) on Day 15 of the estrous cycle as compared with non-RB cows (n = 4). Table S2. List of up- and down-regulated genes in ICAR of ipsilateral uterine horns of RB cows on Day 15 of the estrous cycle as compared with non-RB cows. Table S3. List of up- and down-regulated genes in CAR of contralateral uterine horns of RB cows on Day 15 of the estrous cycle as compared with non-RB cows. Table S4. List of up- and down-regulated genes in ICAR of contralateral uterine horns of RB cows on Day 15 of the estrous cycle as compared with non-RB cows. (XLSX 254 kb

Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

<div><p>We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.</p></div

Gel analysis of RNA-primed MPRCA products to detect the contaminating DNA in Phi29 DNA polymerase.

<p>(<b>A</b>) DNA amplification with Phi29 DNA polymerase prepared by our procedure. (<b>B</b>) DNA amplification with commercially available Phi29 DNA polymerase (Epicentre, Lot No. 10710). (<b>C</b>) DNA amplification with Phi29 DNA polymerase prepared by DNase-treatment. All gel images show the <i>Bam</i>HI/<i>Eco</i>RI double digested amplification products. Black arrow indicates the specific amplification products from pUC19; M, AccuRuler 1-kb DNA RTU ladder (Maestrogen). All reactions were repeated three times or more; typical results are shown.</p

Isolation and purification of Phi29 DNA polymerase.

<p>(<b>A</b>) SDS-PAGE analysis of expressed and isolated GST-ø29pol, and purified Phi29 DNA polymerase. Lane; M, molecular mass markers (Bio-Rad); 1, whole cell lysate; 2, supernatant after PEI precipitation; 3, supernatant after dilution; 4, GS4B resin before digestion by PreScission; 5, GS4B resin after digestion by PreScission; 6, purified Phi29 DNA polymerase (before addition of glycerol); 7, purified Phi29 DNA polymerase (after addition of glycerol); 8, commercially available Phi29 DNA polymerase (1 µg, Epicentre, Lot No. RPH-61004). The arrowhead indicates the GST-ø29pol (calculated size, approximately 94 kDa) and the arrow indicates the purified Phi29 DNA polymerase and commercially available polymerase (calculated size, approximately 67 kDa). (<b>B</b>) Agarose gel analysis of the removal of host DNA by PEI precipitation. Lane; M, AccuRuler 1-kb DNA ladder (Maestrogen); 1, whole cell lysate; 2, supernatant after PEI precipitation.</p

Schematic diagram of the DNA decontamination of Phi29 DNA polymerase.

<p>GST-fused Phi29 DNA polymerase (GST-ø29pol) is expressed in <i>E. coli</i> and affinity purified by glutathione sepharose 4B resin (GS4B) of the cell lysate after polyethylenimine (PEI) precipitation to remove the host DNA. Then, GST-ø29pol is treated with ethidium monoazide (EMA) and irradiated with visible light (VL) to reduce both ssDNA and dsDNA, which may be captured in GST-ø29pol. After washing away the free EMA, the remaining oligonucleotides are reduced by the endogenous 3′-5′ exonuclease activity of the polymerase. Finally, DNA-free Phi29 DNA polymerase is separated from GS4B by digestion with PreScission protease and collected.</p