Detection of mutations by RNAse cleavage

Principle

This method is based on the fact that many ribonucleases will cleave single stranded RNA and therefore will cleave DNA:RNA or RNA:RNA hybrids in heteroduplexes where there is a mismatched base. Cleavage of RNA is thought to occur 3’ to pyrimidines and of the 12 possible types of mismatches, four are recognized more efficiently (C:A, C:C, C:T, U:T). The sequence surrounding the mismatch may be important in determining the efficiency of cleavage.

There are four chances of detecting mutations by this method because when heteroduplexes are formed, 2 complementary duplexes are present containing four mismatched bases:

WT	MUT	HET1	HET2
G	A	G	A
C	T	T	C

All that is required then is for only one of these to be cleaved to allow mutation detection.

1. 32P labelled RNA probes of both senses are synthesized from a wild type DNA template using the SP6 transcription system. The DNA template can either be a restriction fragment or PCR amplified DNA.
2. The RNA probe is then hybridised to denatured test DNA in solution.
3. The RNA:DNA hybrid mixture is treated with RNAse and then phenol-chloroform extracted/ethanol precipitated.
4. The products are analysed by electrophoresis in a standard sequencing gel followed by autoradiography.
5. If the test DNA is identical to the wild-type DNA then a single band is seen. If the test DNA contains a single base substitution that results in a mismatch, two new RNA fragments are observed. The total size of the two new fragments should be equal to the size of the RNA of the wild-type DNA.
6. The position of the mutation can be localized relative to the ends of the RNA probe by determining the sizes of the cleavage products. If a second experiment is carried out using DNA which has been digested with an enzyme which cleaves once within the sequence homologous to the probe, an additional fragment will be generated. After hybridisation, each DNA:RNA duplex will contain a single-stranded overhang of RNA probe which will be digested away. Electrophoresis will show the replacement of one of the RNA products by two bands and the position of the mismatch relative to the restriction site can be determined.

1. PCR amplification of wild-type and test DNA. Bacteriophage promoters (T7 and SP6) are incorporated into the 5’ ends of the PCR primers and therefore into the PCR amplified DNA.
2. Transcription of both strands of the wild-type and test PCR product.
3. Sense and anti-sense transcripts are mixed with equal volumes of complementary wild-type transcripts to produce double stranded RNA duplexes. Equal volumes of sense and anti-sense transcripts of wild-type are mixed as the control.
4. RNAse treatment of the duplex RNA.
5. Analysis of cleavage products on 2% agarose gels. No purification prior to gel loading is required.

The NIRCA commercial kit includes a helix modifying reagent that makes the mismatches more sensitive to cleavage.

The position of the mutation can be localized to within a few nucleotides.
Scanning of DNA up to 1.6kb possible.
Can be applied to unamplified genomic DNA.
Deletions readily detected.

Disadvantages

Cannot detect homozygous mutations unless wild-type DNA is added to test DNA.
Only detects 60-80% of point mutations although this can be increased to 80-90% if both strands are screened for mismatches. Additional RNase enzymes such as RNase 1 and RNase T1 increase the sensitivity of the assay further.
It is necessary to prepare template for RNA probe by cloning or PCR.
One slight disadvantage of the NIRCA test is that primers have to be made which include bacteriophage polymerase promoters.

Applications

RNAse cleavage has been used as a mutation scanning technique in APC, OTC, p53, Factor IX, type 1 collagen.

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