Use of Minigenes in a Diagnostic Laboratory. Allan Richards. Cambridge

Transcription

1 Use of Minigenes in a Diagnostic Laboratory Allan Richards Cambridge

2 Minigene Analysis Minigenes are used to assess the effect of sequence variants on the processing of pre-mrna into the final message transcript Also known as splicing reporters Advantage:- No need for tissue from the patient Disadvantage :- Artificial system and may not replicate in vivo effect Used in conjunction with in silico analysis family inheritance and variant databases

3 Minigene Analysis is Included in the National Stickler Syndrome Diagnostic Service for the Collagen Genes COL2A1 and COL11A1. A Exon 9 c a>g S M N B Exon 9 c a>g S M N * 500 bp 500 bp * N +79T +83G C Exon 19 c g>a S N M Exon 38 c _+26del22 S M N D E Exon 42 c t[3] S M N 500 bp 500 bp 500 bp F Exon 51 c g>c S N M G Exon 51 c dupt S M N 500 bp 500 bp COL11A1 splice site mutations Supplementary Figure 2 COL2A1 Intron 48 Mutations Missplicing :- Frameshifts

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5 Minigene Hybrid Minigene Start signal Stop signal Uses the CMV promoter for high expression in human cells Bovine Growth Hormone polyadenylation sequence

6 Design appropriate primers Check the amplified region for restriction sites Add tags with appropriate restriction sites / kozak / start sequence to gene specific primer sequences before ordering. e.g gggggggaattcgacatg EcoR I kozac Start Exon

7 Clone-PCR Worksheet Details lab patient specific DNA bank number Primer / enzyme / amplification conditions. Transfer checks. Additional pages with space to paste photo of PCR product Instructions for restriction enzyme digestion. Quantification of restricted product (Gel photo) Instructions for Ligation Reaction; Ratio Insert:Vector etc

8 A/G

9 6-8 clones picked for DNA Mini-Preps (5ml cultures) Clones are named with first letters of given and family name i.e ThBo 1 and dated Clone DNA mini-preps are sequenced using a Cloneseq worksheet Still using patients unique lab DNA number but annotated with Clone names and date of mini-prep. Transfers are checked. Mutation surveyor projects created for each clone mini-prep to identify normal and variant clones. Projects named with worksheet number_clone name_date_sequencing run Only DNA mini-preps without cloning errors are used. Polymorphisms are OK

10 Cloned DNA mini-preps are used to transfect cultured human cells Along with a vector (Negative) control Initial transfer must check name and date of cloned DNA mini-prep All transfer are checked

11 RNA is prepared 24h after transfection using Qiagen RNeasy kits All transfers are checked RNA is DNAse I treated to remove any contaminating cloned DNA At the final transfer each RNA sample is given a unique Lab number & RNA bank location e.g.

12 Clone-RT-PCR worksheet is created using the unique Lab RNA sample numbers RT reaction is performed using a vector specific primer PCR reaction is performed using a vector and construct specific primer Eliminating the possibility of amplifying endogenous RNA Products are analysed by gel electrophoresis and sequencing V N C Mutation surveyor project created using either a cdna reference sequence, or by directly comparing one cdna against the other

13 Checking The sequence of the cloned DNA mini-prep (including date) used in the transfections. All transfers from transfection through to RT-PCR product sequencing. The analysis of the RT-PCR mutation surveyor sequencing project.

14 The report is for the result of the minigene analysis (not patient specific) Includes the proviso that the effect may differ in vivo or in other cell types

15 IVS51 Donor Splice site Mutations G+1>T = Stickler syndrome G+1>A = Stickler syndrome T+2>C = Predominantly Ocular Stickler syndrome TGgtaag Only the T+2>C mutation can be spliced normally, but not in all cell lines tested a c t Transfection into various cell types Normal Transcript Specific RT-PCR Eye MIO-M1, Cornea, Sclera Skeletal OUMS27, Saos-2, SW1353

16 N -21bp? Kn1 Kn2 N M N M S 4 00bp 300bp CMV T7 pcdna3.1a S 400bp 300bp Exon 13 Exon 15 ATG AGGCGGGT>AGgtgggt Kn1 Kn2 Normal -21bp Exon Skip Normal -21bp Exon Skip Exon skip Exon skip

17 Acknowledgements Cambridge NHS Molecular lab David Hill :- lab work Genetic Technologists Clinical Scientists Stickler Syndrome Diagnostic Service Martin Snead Annie McNinch