Editing Complementary DNA (section)

==== Second-strand synthesis ====
The result of first-strand syntheses, RNA-DNA hybrids, can be processed through multiple second-strand synthesis methods or processed directly in downstream assays.<ref>{{Cite web |last=Invitrogen |title=cDNA Synthesis System |url=http://tools.thermofisher.com/content/sfs/manuals/18267.pdf |url-status=live |archive-url=https://web.archive.org/web/20181222122300/http://tools.thermofisher.com/content/sfs/manuals/18267.pdf |archive-date=2018-12-22 |access-date=27 May 2020 |website=Thermofisher}}</ref><ref>{{Cite journal |last=Agarwal |first=Saurabh |last2=Macfarlan |first2=Todd S. |last3=Sartor |first3=Maureen A. |last4=Iwase |first4=Shigeki |date=21 January 2015 |title=Sequencing of first-strand cDNA library reveals full-length transcriptomes |journal=Nature Communications |language=en |volume=6 |issue=1 |page=6002 |bibcode=2015NatCo...6.6002A |doi=10.1038/ncomms7002 |issn=2041-1723 |pmc=5054741 |pmid=25607527}}</ref> An early method known as hairpin-primed synthesis relied on hairpin formation on the 3' end of the first-strand cDNA to prime second-strand synthesis. However, priming is random and hairpin hydrolysis leads to loss of information. The Gubler and Hoffman Procedure uses E. Coli RNase H to nick mRNA that is replaced with E. Coli [[DNA polymerase I|DNA Polymerase]] I and sealed with E. Coli [[DNA ligase|DNA Ligase]]. An optimization of this procedure relies on low RNase H activity of M-MLV to nick mRNA with remaining RNA later removed by adding RNase H after DNA Polymerase translation of the second-strand cDNA. This prevents lost sequence information at the 5' end of the mRNA.