Selección de los componentes del sistema hidráulico

COSEGREGATES WITH RCD-1

4.1 Introduction

Before the exon/intron mapping, intron sequence characterisation and subsequent complete screening of all pdeb exons could be carried out, a mutation was communicated in GPRA-affected Irish setters in the USA (W. Baehr, personal communication). This

information was later published in a full paper (Suber et al, 1993) following correction of

their cDNA sequence upon comparison with the one obtained during this project. The dogs

had an identical phenotype {rcd-1) to the setters maintained in a breeding colony at the A HT,

whose genomic DNA had been extracted to be used for screening. Therefore, this genomic DNA was used as a template for PGR amplification and sequencing which confirmed the presence of this mutation in affected Irish setters.

In addition, a rapid, non-isotopic diagnostic test for this mutation was developed, in order that individuals could be screened using the relatively non-invasive technique of blood sampling. This diagnostic test was used to show cosegregation of the mutant allele in the A HT breeding colony. The possible effects of this mutation are discussed.

4.2 A pdeb nonsense mutation is present in rcd-1 affected dogs

Suber and coworkers (1993) reported a G to A transition at codon 807 of pdeb in -affected Irish setters which is an amber stop mutation. The position of this Trp307X mutation is shown in figure 3.19. In order to screen affected and carrier dogs from the different A HT breeding colonies, primers W 100 and X21R (see figure 3.19 for the position and sequence) were used to amplify a fragment of DNA corresponding to exon 21 of pdeb. Figure 4.1 shows that the expected fragment size of about 130bp was obtained for each individual. The remaining 80% of each reaction was purified using NA-45 DEAE paper

(section 2.6.3.1) and resuspended in \Opt\ of SOW. The entire contents were then directly

sequenced using primer X2IR which had been end-labelled as in sections 2.8.3.2 and 2.8.3.3.

A f v l i ' h r n u l u l i n n c o s c u r c ^ a l e s w i t h r e d - 1

2 7 0 B P - 1 3 0 B P -

Figure 4.1 Agarose gel of an exon 21 PCR from a genomic DNA template prior to direct sequencing. The rcd-1 affected and carrier animal samples were from the AfFF colony of Irish setters. M = PhiX174/HaellI markers (Promega), N = normal dog, C = carrier dog, A = affected dog. A l l c c t c i l K \ i 1 I c U I ( a r r i c r u\l 1 +- G A T C G A T C S T R A N D + - 5' 3' T A A T E G C 3' 5‘ » f f

' #

$*

$ S T R A N D + 3' 5* A T TC AG C G 5' 3" ( ( ) D ( > N A . M I N O A C I D T C C. I K Y P i O i ’l l A N 1 A C. Slop

Figure 4.2 Section of the antisense strand of two of the directly sequenced exon 21 K R prcxiucts generated in figure 4.1 There is a G to A transition depicted which is present in the homozygous state in the rcd-1 affected animal and the heterozygous state in the carrier dog I'he normal sequence at this point contains a G nucleotide. I'he theoretical amino acid translation is given beneath showing that this transition creates a stop c(xlon. G = guanine, A = adenine, 1 = thymidine, C = cytosine.

A p d e b mutation cosegregates with rcd-1

The resulting termination fragments were visualised by denaturing polyacrylamide gel electrophoresis (section 2.8.4).

Figure 4.2 shows a portion of the sequencing gel spanning the putative mutation site for rcd-llrcd-1 and rcd-H+ dogs. There is clearly a single nucleotide substitution which is present in the homozygous state in the affected dog, but only in the heterozygous state in the carrier. The alternative base shown in the carrier is identical to that of a normal dog (data not shown but see figure 3.19). If the positive strand sequence is examined by taking the complement of that shown in the autoradiogram, the result shows that the presence of the G to A transition reported by Suber and coworkers (1993) is confirmed in affected Irish setters in the UK. The alteration of codon 807 from TGG to TAG causes the theoretical Trp807X in the protein.

DNA from a GPRA-affected Tibetan terrier (TT) and miniature longhaired dachshund (MLHD) also kept in breeding colonies at the A HT was PCR-amplified, purified and sequenced in the same way. The nucleotide composition at codon 807 is the same as normal dogs, indicating that the disease in these breeds is not caused by the same mutation as

it is in the rcd-1 Irish setters, a fact previously unestablished.

4.3 Development of a diagnostic test

A search of the MacVector version 3.5 (see section 2.9.1) restriction site database revealed that the G to A transition neither creates or deletes a restriction endonuclease recognition site. Therefore, there are no existing restriction fragment length polymorphisms for which to test. In order to generate an allele-specific, unique restriction site which would differentiate between the normal and mutant allele, a special PCR primer had to be designed.

4.3.1 Design of a PCR primer to create a restriction endonuclease site

A manual search of a restriction enzyme database from the above software (MacVector version 3.5) revealed that the isoschzomers M ael/Bfal/Rm al have a recognition site, CTAG which is only one base pair different from that of the pdeb sequence in the vicinity of the mutation. Figure 4.3 shows how this primer (X21Fmis, whose position is also

A p d eb mutation cosegregates with rcd-1

GENOMIC DNA

114 bp

91 bp

RESIDUE 807

5 ’

EXON 21

primer E 2 1 F

_{primer E 21 R}

5 '-G A a G C A G A A C A A C A G G A A æ A A ™ A A G ..r3 ’ NORM AL

5'- GACTGCAGAACAACAGGAAGGAATAGAAG..r3' MUTANT

5'- GACTGCAGAACAACAGGAAGGACT-3'

Î

primer E 21 F

MISMATCH

Bfa I

I

S'.GACTGCAGAACAACAGGAAGGACTAGAAG...-3'

Figure 4.3 Schematic diagram of the strategy of the rcd-1 diagnostic test. The mismatch between primer X21 Finis and genomic DNA creates a sequence in the PCR product (generated from a genomic DNA template) which will only cut with Bfal if the rcd-1 G to A transversion is present.

A p d e b mutation cosegregates with rcd-1

given in figure 3.19) should amplify genomic DNA in combination with primer X21R, under conditions which allow for the mismatched sequence to aimeal. The amplified product will contain a unique Bfal site only if the mutant allele is amplified and not for the normal allele. PCR-dependent Bfal diagnosis can then be visualised by ethidium bromide staining, since the amplification procedure generates a large amount of product.

4.3.2 Amplification and Bfal digestion of the 'mismatched' exon 21

The estimated dissociation temperature of primer X21Fmis is 72°C by eye and 62PC

using Geneworks software (sections 2.3.1 and 2.9.1). An intermediate annealing temperature

of 64®C was initially used on the genomic DNA in a lOOjA, 40-cycle PCR. A clean band was

obtained in a I0j4\ aliquot of the reaction for each individual. To maximise the yield, the PCR

was repeated in 100//1 volumes using identical conditions but the template used was a 2]a\

aliquot of LOT agarose-purified W100/X21R band amplified from each individual. The reactions were used as a stock for initial Bfal digestion experiments.

These experiments, using 5U of enzyme and incubated overnight showed that the enzyme did not appear to cut when the PCR reaction was used directly in the digest, along

with the manufacturer's recommended buffer whereas a K template was completely digested in

a separate reaction under identical conditions, except for the absence of the PCR reagents and buffer (data not shown). The PCR product was therefore purified from the reaction constituents by ethanol precipitation (section 2.6.1). Aliquots of 5//1 were used in overnight digests at 37®C.

Despite this purification step, the source of the DNA and/or the quality of the particular batch of enzyme appeared to dramatically alter the effectiveness of the digestion. Figure 4.4 shows template generated directly from genomic DNA which has digested incompletely after a 20 hour incubation. In order to achieve complete digestion, the reaction was spiked with an additional 5U of Bfal and reincubated on a further two occasions before a satisfactorily complete digest shown in figure 4.5 was obtained. This result shows that the diagnostic test clearly detects all three allele combinations when the digest is complete. Polyacrylamide gel electrophoresis was the size fractionation method of choice since it gives a

f-'igurc 4 .4 P(4yacr\'laniidc gel ol tlic cxon 21 d ia g n o stic lest PCR products generated directly Iroin g en o m ic D N A and d igested tor 20 hours w ith R ial. M = PhiX 174/H aelII markers, N = n o n n a l d og, C = carrier dog, A = a llected dog.

A jn i e h n t u la li o n ( wi t h r e d 1

In document Diseño y construcción de una descortezadora de coco (página 77-87)