Use of Minigenes in a Diagnostic Laboratory

Allan Richards

Cambridge

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

Exon 51 c.3816+2dupT

S M N

Exon 19 c.1845+5G>A S N M

Exon 51 c.3816+5G>CS N M

Exon 42 c.3168+2T[3] S M N

Exon 9 c.991-24A>G

S M NExon 9 c.991-24A>G S M N

500 bp 500 bp

500 bp500 bp

500 bp

500 bp500 bp

A

C E

GF

Exon 38 c.2862+5_+26del22 S M N

D

B

**

Supplementary Figure 2

Minigene Analysis is Included in the National Stickler Syndrome Diagnostic Service

for the Collagen Genes COL2A1 and COL11A1.

N +79T +83G

COL2A1 Intron 48 Mutations

Missplicing :- Frameshifts

COL11A1 splice site mutations

Minigene Hybrid Minigene

Uses the CMV promoter for high expression in human cells

Bovine Growth Hormone polyadenylation sequence

Start signal Stop signal

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

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

A/G 1 2 3 4 5 6

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

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

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.

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

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.

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

IVS51 Donor Splice site Mutations

G+1>T = Stickler syndrome

G+1>A = Stickler syndrome

T+2>C = Predominantly Ocular

Stickler syndrome

Only the T+2>C mutation can be spliced

normally, but not in all cell lines tested

TGgtaag

a c

t

Normal Transcript

Specific RT-PCR

Transfection into various cell types

Eye Skeletal MIO-M1, Cornea, Sclera OUMS27, Saos-2, SW1353

S

Kn1 Kn2

N M N M S

300bp

300bp

400bp

4 00bp

Exon 13 Exon 15

N

?

-21bp

Kn1 Kn2

Exon skip Exon skip

Normal -21bp Exon Skip

Normal -21bp Exon Skip

13 14 15 16 ATG

AGGCGGGT>AGgtgggt

pcDNA3.1A

CMV T7

Acknowledgements

Cambridge NHS Molecular lab

David Hill :- lab work

Genetic Technologists

Clinical Scientists

Stickler Syndrome Diagnostic Service

Martin Snead

Annie McNinch