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Purpose:
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Denaturing gradient gels are used to detect non-RFLP polymorphisms. The small (200-700 bp) genomic restriction fragments are run on a low to high denaturant gradient acrylamide gel; initially the fragments move according to molecular weight, but as they progress into higher denaturing conditions, each (depending on its sequence composition) reaches a point where the DNA begins to melt. The partial melting severely retards the progress of the molecule in the gel, and a mobility shift is observed. It is the mobility shift which can differ for slightly different sequences (depending on the sequence, as little as a single bp change can cause a mobility shift). Alleles are detected by differences in mobility.
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Principle
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Denaturing gradient gel electrophoresis has been shown to detect differences in the melting behavior of small DNA fragments (200-700 bp) that differ by as little as a single base substitution. When a DNA fragment is subjected to an increasingly denaturing physical environment, it partially melts. As the denaturing conditions become more extreme, the partially melted fragment completely dissociates into single strands. Rather than partially melting in a continuous zipper-like manner, most fragments melt in a step-wise process. Discrete portions or domains of the fragment suddenly become single-stranded within a very narrow range of denaturing conditions. The rate of mobility of DNA fragments in acrylamide gels changes as a consequence of the physical shape of the fragment. Partially melted fragments migrate much more slowly during electrophoresis through the polyacrylamide matrix than completely double-stranded fragments. When a double-stranded fragment is electrophoresed into a gradient of increasingly denaturing conditions, it partially melts and undergoes a sharp reduction in mobility because it changes shape. In practice, the denaturants used are heat (a constant temperature of 60 degrees C) and a fixed ratio of formamide (ranging from 0-40%) and urea (ranging from 0-7 M). The position in the gradient where a domain of a DNA fragment melts and thus nearly stops migrating is dependent on the nucleotide sequence in the melted region. Sequence differences in otherwise identical fragments often cause them to partially melt at different positions in the gradient and therefore 'stop' at different positions in the gel. By comparing the melting behavior of the polymorphic DNA fragments side-by side on denaturing gradient gels, it is possible to detect fragments that have mutations in the first melting domain.
Many fragments can be analyzed simultaneously on a single denaturing gel in which the direction of electrophoresis is perpendicular to that of the denaturing gradient. When a large number of different fragments is electrophoresed, the fragments can be identified by their molecular weight in the low denaturant side of the gel. By following the S-shaped curves, the characteristic denaturant concentration at which the first domain melts can be determined. When two nearly identical sets of fragments are mixed together and electrophoresed into a 'perpendicular' denaturing gradient gel, the melted domains that have sequence differences between each other will melt at slightly different positions and produce double bands.
Sequence differences are often easily detected in DNA fragments when nearly identical digests are electrophoresed in the same direction as that of the denaturing gradient. These 'parallel' gels permit the simultaneous comparison of as many sets of fragments as there are lanes on the gel, unlike the perpendicular gels. The procedures below refer almost entirely to parallel denaturing gradient gels.
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DGGE blotting
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Denaturing gradient gel blots of genomic DNA can be used to detect most single base differences (which may occur as frequently as every 400 bp). Genomic DNA is digested with an enzyme combination that cuts the DNA into fragments of average size 200-700 bp. Enzymes with 4-base recognition sites, such as AluI, HaeIII, HhaI, MspI, and RsaI are the most useful. The samples are electrophoresed long enough for many fragments to reach a position in the gradient at which the first melting domain denatures. After electrophoresis, the DNA fragments are transferred to a nylon blot. The blots are hybridized overnight with a radioactive probe, washed, and exposed to X-ray film for 1-5 days. Each lane is examined for fragments that have mobility shifts.
Many polymorphisms are present in portions of a restriction fragment not in the first melting domain. Sequence differences are undetectable unless they are within the first melting domain. To avoid this problem, it is often possible to 'move' the mutation into the first melting domain by using different restriction enzyme combinations. Frequently, at least one of 4-5 different digests achieves this result. Occasionally, a mobility shift is observed in more than one restriction digest. By aligning on a restriction map the partially overlapping fragments that have altered melting behavior, the region containing the the base di Detection of mutations by RNAse cleavage
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Principle
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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:
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WT
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MUT |
HET1
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HET2 |
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G |
A |
G |
A |
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C |
T |
T |
C |
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All that is required then is for only one of these to be cleaved to allow mutation detection.
Method
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Radioactive |
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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.
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The RNA probe is then hybridised to denatured test DNA in solution.
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The RNA:DNA hybrid mixture is treated with RNAse and then phenol-chloroform extracted/ethanol precipitated.
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The products are analysed by electrophoresis in a standard sequencing gel followed by autoradiography.
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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.
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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.
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Non-radioactive |
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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. |
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Transcription of both strands of the wild-type and test PCR product. |
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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. |
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RNAse treatment of the duplex RNA. |
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Analysis of cleavage products on 2% agarose gels. No purification prior to gel loading is required. |
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The NIRCA commercial kit includes a helix modifying reagent that makes the mismatches more sensitive to cleavage.
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Advantages
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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.
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Disadvantages
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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.
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Applications
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RNAse cleavage has been used as a mutation scanning technique in APC, OTC, p53, Factor IX, type 1 collagen.
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References:
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Goldrick MM, Kimball GR, Liu Q, Martin LA, Sommer SS, Tseng JYH. NIRCA(Tm) - A Rapid Robust Method For Screening For Unknown Point Mutations. (1996) Biotechniques 21:106-12.
Grange DK, Gottesman GS, Lewis MB, Marini JC. Detection Of Point Mutations In Type-I Collagen By RNase Digestion Of RNA RNA Hybrids. (1990) Nucleic Acids Research 18:4227-36.
Miyoshi Y, Nagase H, Ando H, Horii A, Ichii S, Nakatsuru S, Aoki T, Miki Y, Mori T, Nakamura Y. Somatic mutations of the APC gene in colorectal tumors: mutation cluster region in the APC gene. (1992) Hum Mol Genet 1(4):229-33.
Mutation Detection (OUP, 1997) Cotton, R.G.H.
Myers RM, Larin Z, Maniatis T. Detection Of Single Base Substitutions By Ribonuclease Cleavage At Mismatches In RNA-DNA Duplexes. (1985) Science 230:1242-6.
NIRCA: A rapid robust method for screening for unknown point mutations. (1996) Biotechniques 21:106-112.
Taylor and Deeble. Enzymatic methods for mutation scanning. (1999) Genet Anal 14(5-6);181-6.
Taylor. Enzymatic and chemical cleavage methods. (1999) Electrophoresis 20(6);1125-30.
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