S1 Nuclease Mapping

•S1 endonuclease (from the fungus Aspergillus oryzae) only cleaves single strand.•Nuclease S1 will digest only ssDNA or ssRNA.

S1 Nuclease mapping

• Allow the precise start and end points of the transcript and of any introns it contains to be mapped onto the DNA sequence.

• Transcript mapping• S1 nuclease mapping is used to locate the start point of a

transcript.• If one of the strands is labeled at one end, the length

of labeled fragment remaining after hybridization and nuclease digestion reflects the point on the probe where the two sequences diverge

• This is the basis for S-1 mapping of transcriptional start sites

A probe is chosen that is complementary to the RNA, and extends past the anticipated start site. The 5' end is 32P labeled, and chosen to fall within the coding region of the mRNA, so that it will be protected from digestion. After hybridization, the 3' overhang of the probe is digested away, and the size of the remainder of the probe is accurately determined on a denaturing PAGE Gel. The distance between the known labeling site and the new end of the probe gives the transcriptional start site to within 1 base

Locate the start point of a transcript

Locate the end point of a transcript

S1 mapping of an intron site

S1 mapping of an intron site

S1 mapping can also be used to find intron sites .In this case, the probe is derived from genomic DNA, and again labeled so that the labeled 3' end falls within a coding portion of the gene. Any intron in this construct will not find a homologous region in the RNA, and will be cleaved by the S-1 nuclease. In this case, the size of the labeled remainder reflects the distance from the label site to the splice site.

Here, a Sau3A fragment that contains 100 bp of coding region, along with 300 bp of the leader sequence preceding the gene, has been cloned into an M13 vector and obtained as a single-stranded molecule. A sample of the RNA transcript is added and allowed to anneal to the DNA molecule. The DNA molecule is still primarily single-stranded but now has a small region protected by the RNA transcript. All but this protected region is digested by S1 nuclease and the RNA is degraded with alkali, leaving a short single-stranded DNA fragment. If thesemanipulations are examined closely it will become clear that the size of this singlestranded fragment corresponds to the distance between the transcription start point and the right-hand Sau3A site. The size of the single-stranded fragment is therefore determined by gel electrophoresis and this information is used to locate the transcription start point on the DNA sequence. Exactly the same strategy could locate the end point of transcription and the junction points between introns and exons. The only difference would be the position of the restriction site chosen to delimit one end of the protected single-stranded DNA fragment

Locating a transcription start point by S1 nuclease mapping

S1 Mapping of RNA