Single-strand binding proteins

Nat Commun. 2018 Feb 12;9(1):619. doi:10.1038/s41467-018-02866-0

Hayashi T, Ozaki H, Sasagawa Y, Umeda M, Danno H, Nikaido I.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

RamDA-seq. First-strand cDNAs are synthesised with a RNAse H minus reverse-transcriptase. DNAse I introduces nicks that prime synthesis of new cDNA molecules. T4gp32 promotes the strand-displacement activity of the reverse-transcriptase. Second-strand cDNAs are synthethised with Klenow fragment (3′ → 5′ exo-) primed with NSRs. In single cells, genomic DNA needs to be removed because of the DNAseI digestion during RT and the low-complexity priming of the second-strand synthesis with NSRs. The resulting DNA molecules are tagmented and sequenced with standard methods. Thus, the method is non-stranded. Despite the use of NSRs, the rRNA rate stays between 20 and 30 %.

Inoue J, Shigemori Y, Mikawa T.

Nucleic Acids Res. 2006 May 17;34(9):e69 doi:10.1093/nar/gkl350

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein.

By weakening the DNA-protein interaction, the authors acheived the suppression of the non-temlated products in rolling circle amplification.

Nucleic Acids Res. 2001 Mar 1;29(5):E29

Baugh LR, Hill AA, Brown EL, Hunter CP.

Quantitative analysis of mRNA amplification by in vitro transcription.

T7 pol generates aberrant template-unrelated products. This is fixed by reducing the concentration of oligo dT primers, and of enzymes. Under these conditions, 2 rounds of RNA linear amplification is not generatng too much bias. Small-volume protocol. T4gp32, a single stranded protein, increases RT processivity.

J Biomol Tech. 2005 Sep;16(3):239-47

Piché C, Schernthaner JP.

Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein.

T4gp32 increases the yield of the smallest cDNAs by 10 %.

Kenzelmann M, Klären R, Hergenhahn M, Bonrouhi M, Gröne HJ, Schmid W, Schütz G.

Genomics. 2004 Apr;83(4):550-8 doi:10.1016/j.ygeno.2003.09.026

High-accuracy amplification of nanogram total RNA amounts for gene profiling.

Augment temperature, lower primer concentration, and add T4gp32 to enhance reverse transcription.