Commentary - Research and Reports in Immunology (2023) Volume 6, Issue 8
Moloney Murine Leukaemia Virus (MMLV)-based reverse transcriptase ads non-templates and switches templates at the same time, competing with each other.
Department of Immunology, The University of Hong Kong, Kowloon, Hong Kong, China
- Corresponding Author:
- Wing Chan
Department of Immunology
The University of Hong Kong
Received: 03-May-2022, Manuscript No. AARRI-22-62665; Editor assigned: 06-May-2022, AARRI-22-62665 (PQ); Reviewed: 20-May-2022, QC No. AARRI-22-62665; Revised: 01-Jul-2022, Manuscript No. AARRI-22-62665 Published: 08-Jul-2022, DOI: 10.35841/aarri-5.4.117
Citation: Chan W. Moloney Murine Leukaemia Virus (MMLV)-based reverse transcriptase ads non-templates and switches templates at the same time, competing with each other. Res Rep Immunol. 2022;5(4):1-2.
Single-cell RNASeq (scRNA-Seq) provided an unprecedented understanding of gene expression and regulation single cell. Many scRNA-Seq approaches Moloney Murine Leukemia Virus template switching characteristics (MMLV) type reverse transcriptase. Template switching it has three untemplated prominent nucleotides (CCCs) 3 end of developmental cDNA. Matches the rGrGrG 3 end of the Template Switching Oligo (TSO), Reverse Transcriptase (RT) switches templates Proceed to copy the TSO sequence. In this study Detailed analysis of template switching bias related to depending on the role of RNA template, especially sequence and type 5 ends of these distortions (with and without caps). Our result confirmed the existence of 5 m7 G-Cap improves submissions switching efficiency. Also profile the composition of untemplated additives in the absence of TSO, The 5'end of the RNA template affects terminal transferase activity RT In addition, the design of a new power transmission system operator combined with the most common additions without templates, it was of little use Improved template switching efficiency. Our results, contrary to current understanding, Matrix switching processes, non-matrix addition, and matrix switching are concurrency and concurrency processes .
Moloney Murine Leukemia Virus Reverse Transcriptase (MMLVRT) is widely used for cDNA preparation and cloning. Previous reports have shown that MMLVRT has a strong activity to add a 3'overhang to the end of double-stranded DNA. The activity is unique in two ways. First, MMLVRT can add A, C, G or T but known DNA polymerases preferentially add an overhangs and do not efficiently add other nucleotides 7-10. Second, it is so active that DNA molecules attach almost completely with an overhang of 1-5 nucleotides in a single reaction . This activity is potentially useful because the high tailing efficiency allows the adapter DNA molecule to ligate to each DNA molecule in reaction. This allows for a thorough analysis of the DNA pool. In addition, it reduces the amount of DNA required for analysis and enhances single cell genomics or other analysis of rare DNA from fossil or ecosystem sources. We also showed that dAMP, dCMP, and dGMP specifically enhance T, G, and C tailing, respectively. In this study, we searched for additional compounds that enhance MMLVRT tailing activity and found that adenine, cytosine, and guanine and their cognate nucleotides enhance T, G, and C tailing, respectively .
In this study, the strong tailing activity of MMLVRT6 was further characterized by identifying additional enhancing compounds. The molecular mechanism involved in the tailing enhancement could be the transient Watson Click base pairing formed between an enhancer molecule and the deoxy ribonucleotide triphosphate molecule to be incorporated. We propose that an enhancer molecule binds to the active center of MMLVRT in an orientation similar to that taken by a single ribonucleotide moiety in an RNA molecule in a revers transcription reaction. Of note, bases are not efficient enhancers, suggesting that the base moiety and the ribose or deoxyribose moiety of the compounds are involved in the binding . Although we have tested different compounds, other nucleotide analogues, such as cyclic AMP, NADH, and cyclic di GMP, or the wealth of compounds developed for anticancer therapy, might have even stronger effects, and the 3D structure of MMLVRT11 and molecular docking approaches may help the search for better tailing enhancers. Compounds designed to form a Watson Crick base pair with dATP have been tested, but none have enhanced tailing. The base stacking interaction 7 between dATP and the base of deoxyribose or DNA tail is so strong that the two hydrogen bonds provided by the enhancer molecule may not promote the tailing reaction. In fact, the unenhanced dATP is the fastest built-in dNTP, adding 3-5 asses within 10 minutes .
Continuous nucleotide addition by MMLVRT in the presence of GMP, GDP, and CMP is a technical application such as DNA terminal labelling, TUNEL assay, RACE, homopolymer tail-mediated ligation PCR, as well as terminal nucleotidyltransferase. May be useful for. The difference in the magnitude of the enhancer effect for the various tailing responses described in this study provides the basis for leveraging MMLVRT template-independent tailing activities. Its practical application requires further improvements for specific applications, but this activity is associated with nextgeneration sequencing, conventional TA or CG cloning, and adapter molecules at the DNA ends in cosmid library preparation. It can be used for efficient ligation.
- Kapteyn J, He R, McDowell ET, et al. Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples. BMC genomics. 2010;11:413.
- Zhou MY, Gomez-Sanchez CE. Universal TA cloning. Curr Issues Mol Biol. 2000;2(1):1–7.
- Shinnick TM, Lerner RA, Sutcliffe JG, et al. Nucleotide sequence of Moloney murine leukaemia virus.Nature. 1981;293(5833):543–548.
- Schwarzschild MA, Dauer WT, Lewis SE, et al. Leukemia inhibitory factor and ciliary neurotrophic factor increase activated Ras in a neuroblastoma cell line and in sympathetic neuron cultures. J. Neurochem. 1994;63(4):1246–1254.
- Roche S, McGlade J, Jones M, et al. Requirement of phospholipase C gamma, the tyrosine phosphatase Syp and the adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence for the existence of Ras-dependent and Ras-independent pathways. EMBO J. 1996;15(18):4940–4948.