ANÁLISIS HTTP PARA LA CONSTRUCCÓN DEL PROTOTIPO DE

5' GAG CGG GGA CTT TGT CTC CT 3' and 5 ' CTT TGA CCT GAC TGA CGA T 3' Expected size of PCR product: 700bp. Human DMAs were used as positive

controls.

5) STS from 33CA11/Left end clone

5' ACT ACG GAA TTC CCA CTT 3' and 5' AAC TTA CTT GGT CTC TTG 3'. Expected size of PCR product: lllb p .

6) STS from 33CA11/Right end clone Described in the previous section. 7) STS from 20CC8/Left end clone

5' AGG CCA GGT GCA GTG ACT CAT G 3' and 5' TTG CCA TGT TGC CCA GGC TGG 3'. Expected size of PCR product: 107bp

8) STS from 20CC8/Right end clone

5' CTG ATG CCA CTG AGT TAT AGG 3' and 5' CTT AGG TAA ATA TTC TGG GGC C 3'. Expected size of PCR product size: 1 0 1 bp.

2.2 Isolation of YAC ends using the Vectorette svstem

2.2.1 YAC33CA11

Two libraries were constructed (as described in section 4.6.1 of chapter 2) using the enzymes Rsal and Haelll. PCR amplification of the Rsal library was carried out for the left end of the clone and of both libraries for the right end of the clone. Protocols were as described in section 4.6 of chapter 2. During PCR amplification, the

Universal Vectorette primer was unphosphorylated and the vector arm primers phosphorylated. PCR products were analysed on 1% agarose gel. They were then partially purified by phenol extraction and converted into single stranded DNA using

the Lambda exonuclease method (4.7.1 a, chapter 2). The products were

sequenced using the end labelled left and right vector arms sequencing primers (4.7.2 a).

2.2.2 YAC 20CC8

Two libraries were constructed (section 4.6.1, chapter 2) using the enzymes Rsal and EcoRV. Protocols for PCR amplification of the two libraries were modified. PCR amplifications were carried out for 40 cycles of dénaturation at 94°C for 1 minute, annealing at 62° (or at 65°C) for 1 minute and extension at 72°C for 3 minutes. PCR products were analysed on a 2.5% agarose gel. Components in the PCR reactions, size of PCR products and sequencing method were as follows:

1) Right arm of the YAC

PCR components: 5pl of Vectorette library, lOpI of lOx PCR buffer, lOOpM of dNTPs, 1|xM of phosphorylated pYAC4 right arm primer, IpM unphosphorylated Universal Vectorette primer. PCR products of 400bp and -Ik b were obtained for Rsal and EcoRV libraries, respectively.

2) Left arm of the YAC

PCR components: lO^il of Vectorette library, lOpI of lOx PCR buffer, lOOpM of dNTPs, IpM (Rsa I library) or 0.5^iM (EcoRV library) of phosphorylated pYAC4 left arm primer, IpM (Rsal library) or O.SpM (EcoRV library) unphosphorylated Universal Vectorette primer. PCR products of 500bp and 400bp were obtained for Rsal and EcoRV libraries, respectively.

The Rsal library PCR products were column purified, converted into single stranded DNA using the Lambda exonuclease method (4.7.1 b, chapter 2) and sequenced using YAC arm sequencing primers for °2p incorporation labelling (4.7.2 b, chapter 2). Sufficient sequence data were obtained, therefore EcoRV PCR products were not further analysed.

2.3 Isolation of a (CA)„ repeat from 33CA11 YAC

2.3.1 Isolation o f cosmid clones at the end o f 3 3 C A ll YAC

Following the isolation and sequencing of the end clones of YAC 33CA11, PCR amplification primers were designed and used to amplify 33CA11 YAC DNA and genomic DNA, The PCR products of the right end clone were used to screen the ICRF (Hans Lehrach et al., 1990) reference X-specific cosmid library no. 104 (L4/FCS X). This library was constructed by Dean Nizetic (Nizetic et al., 1991) from digests of DNA from "Flow-sorted" Human X-chromosome and ligated into Lawrist4 vector. The screening was performed (by Dr Fiona Francis) in ICRF laboratories and resulted in six positive clones ( ICRFc104 C096, ICRFc104 D0480, ICRFc104 FI 080, ICRFC104 D01210, ICRFc104 D08214 and ICRFc104 D10158). Bacterial stabs of these cosmid clones were streaked out on LB agar plates supplemented with 50pg/ml kanamycin and incubated at 37°C overnight. A single colony was

picked from each plate and used to inoculate 10ml of sterile LB broth containing 50|xg/ml kanamycin. Following incubation at 37°C with agitation, DNA was isolated and purified using the Magic M i n i p r e p ™ DNA Purification System (Promega)

according to manufacturer's instructions (except that -9ml of culture was used instead of the recommended 1-3ml). DNA was eluted from the purification

Minicolumn in 50|xl TE and an aliquot (5|xl) of each preparation was electrophoresed on a 0.8% agarose gel to check sample quality and estimate DNA concentration. An aliquot (3|xl of 1:1000 dilution) of each cosmid DNA was PCR amplified using the primers of 33CA11/Right end clone to identify true positive clones.

2.3.2 A search for CA repeats In a pool o f cosmids

An aliquot (10-20^1) of each positive cosmid clone was digested with 20units Hindlll in a 25pl reaction containing 2.5^1 of lOx appropriate restriction buffer at 37°C for 2 hours and the entire reaction was electrophoresed on a 0.8% agarose gel alongside X.Hindlll and a lOObp ladder (Gibco BRL) as size markers. The gel was blotted

(using Hybond N+ membrane from Amersham, according to manufacturers' instructions) and the filter hybridised with an oligonucleotide (GT)^i+i probe to identify (CA)^ repeats in the cosmids.

2.3 3 Isolation o f a (CA)„ repeat from a cosmid using the Vectorette system

2.3.3.1 Construction of Vectorette library

A Vectorette library was constructed from cosmid ICRFc104 D01210 using the enzyme Alul as follows: An aliquot (15pl) of cosmid DNA was digested with 20units of Alul (Pharmacia) in a 50|xl reaction volume containing 1x restriction buffer at 37°C for 11/2 hours. Following digestion, 5|xl (Spmol) of the appropriate Vectorette units (CRB), 1 pi of a lOOmM ATP and Sunits of T4 DNA ligase (Pharmacia) were added to the same microtube. The ligation reaction was incubated at 37°C for two hours after which lOOfil of SDW was added and the Vectorette library was stored in small aliquots at -20°C

2.3 3.2 Vectorette PCR

PCR amplification of the Vectorette library was performed using the phosporylated Universal Vectorette Primer (CRB) in combination with either primers C4 (CA)^^ or G4 (GT)ii (both unphosporylated). For PCR amplification 2pl and 5^1 of Vectorette library, lO^il of lOx PCR buffer, 100|liM of each dNTP and IpM (or O.SpM) of each primer were mixed and the volume made up to 99|xl with distilled sterile HgO. The sample was denatured at 95°C for 5 minutes and 2.Sunits of Taq polymerase added at 90°C. PCR conditions were as follows: dénaturation at 94°C for 1 minute,

2.3.3 3 Direct sequencing of the Vectorette PCR products

Single stranded DNA was prepared using the Lambda exonuclease method (section 4.7.1b, chapter 2) and sequenced using the Vectorette sequencing primer (section 4.7.2b, chapter 2).

2.3.3 4 PCR amplification

The primers used for PCR amplification of cosmid or genomic DNA were: 5' TTA GGG TGG GCT TTA ATC TAA S' (CA strand) and 5' GTG TTT CTT GGC TTA CAG ATG S' (GT strand). PCR ampifications were carried out non-radioactively and radioactively as described in section S.4 of chapter B. PCR condtitions for both non­ radioactive and radioactive reactions were as follows: An initial dénaturation at 95°C for 1 minute followed by SO cycles of dénaturation at 94°C for 1 minute, annealing at 56°C (or 58°C) for 1 minute and extension at 72°C for 1 minute. Non-radioactive PCR products were analysed on 2% agarose gels and radioactive products on 6% polyacrylamide sequencing gels.

2.3.4 Verification o f the CA repeat

Following PCR amplification of cosmid (from which the CA repeat derived) and genomic DNA, PCR products were electrophoresed on a 2% agarose gel alongside pBRS22 digested with Avail and EcoRI as size marker. The gel was blotted (using Hybond N+ membrane from Amersham, according to manufacturers' instructions) and the filter hybridised with an oligonucleotide (GT)^^^^ probe to verify the presence of the CA repeat.

3 Results

3.1 Isolation of YACs containing the DXS426 locus

The ICI YAC genomic library was screened by PCR with DXS426. A YAC containing DXS426 locus (DXS426/4, a gift from Dr Mick Coleman ; positive control) and a no

DNA control were included in the screening experiments. Two primary positive pools, 4 and 33, were identified. Secondary pools 4H and 33C were positive. The tertiary pools 7 and 10 for secondary pool 4H, corresponding to clone G2 (see table in section 4.1.3, chapter 2), and pools 1 and 19 for secondary pool 33C,

corresponding to clone A l l, were positive (data not shown). The combined results from the three step screening of the library led to the identification of two positive for DXS426 YAC clones, 4HG2 and 33CA11.

DNA from these clones was prepared in plugs (section 4.3, chapter 2) and

electrophoresed by pulsed field gel electrophoresis (section 4.3, chapter 2) to check quality of the preparation (fig. 26a). Saccharomyces cerevisiae (Strain YNN295 ; Biorad) was used as size marker. The two YACs were not instantly visible but there was indication that they coincided with yeast chromosomal bands. Alkali blotting of the pulsed field gel and hybridisation with total human DNA (sections 4.4 and 4.5, chapter 2) confirmed this, sizing clone 4HG2 at 245kb and clone 33CA11 at 290kb (fig. 26b).

3.2 STS analvsis in YACs 33CA11 and 4HG2

YACs 4HG2 and 33CA11 were analysed for the presence of DXS426, PFC and synapsin loci by PCR. Two YACs containing DXS426 locus (DXS426/1 and DXS426/6, gifts from Dr Mick Coleman) and genomic DNA were used as positive controls. A YAC from a different region was used as a negative control. Both clones contained the DXS426 locus (fig. 27) thus confirming the library screening results. Clone 4HG2 contained PFC whereas 33CA11 did not (fig. 28). Synapsin was absent from both YACs (data not shown).

3.3 Isolation of end clones from YAC 33CA11

Amplification of an Rsal Vectorette library gave single PCR bands of ~0.35kb for the left end and of ~0.3kb for the right end (fig. 29). The pYAC4 left and right arm

Approx. size (kb)

1,125

1,020

M

f ig u r e 26: a. PFGE electrophoresis of 33CA11(lane 2) and 4HG2 (lane 3) YACs. Lane 1 is a YAC clone of known size (245kb). M denotes the Saccharom yces cerevisiae (strain YNN295) size marker.

b. Autoradiograph showing hybridisation of total human DNA to a Southern blot of gel (a). 33C A 11 was sized at 290kb and 4HG2 at 245 kb.