IV. RESULTADOS Y DISCUSIONES
4.1 Análisis microbiológico
4.2.3 Evaluación de color
1.0-1.2kb 0.6kb BamHI CAATATCAAGCTATCCGGATCC- L.H. oligonucleotide 5942 End of known sequence
}
0.5kbFragment lengths after single-stranded PCR
H
I.Okb
H* Hindlll restriction site; Humphries et al, 1984
H^ Hindlll restriction sites; Courtois , personal communication H*+H^ Hindlll restriction sites; Kant et al, 1985
After the first round of PCR, designed to produce single stranded DNA of approximately 0.9kb in length, two distinct main bands were seen for each Hindlll digested DNA sample when run on the agarose gel (figure 3.16a). The fragment sizes were approximately GOObp and 1 .Okb for the individual of genotype T-T- and approximately GOObp and 1.2kb for the individual of genotype T+T+. However, on several repeat PCR's, the two different samples produced bands of the same size, as each other, 700bp and 1.4kb, but different from the first PCR (figure 3.1Gb).
Single stranded PCR of 3' end of a fibrinogen gene Fig. 3.16a 2.0 kb 1.0 kb 0.5 kb T-T- T+T+
Fig. 3.16b
2.0 kb 1.0 kb 0.5 kb T-T- T+T+After the second round of PCR, using the two oligonucleotides, the same bands were observed after electrophoresis as with single-stranded PCR, but they were more intense, indicating that further amplification had taken place. No bands were seen in the "control lane. EcoRI and TaqI restriction digests were performed on both first round PCR product (single-stranded) and second round PCR product (double-stranded) to check for the presence of double-stranded DNA and also a constant EcoRI site and the polymorphic TaqI site. Both the putative single stranded DNA and the double-stranded DNA from the T-T- allele cut with TaqI whereas the DNA from the T+T+ allele did not (figure 3.17).
Figure 3.17
TaqI digested 'single-stranded' PCR of the S' end of the a-fibrinogen gene
ds ss ds
E U T E U T E U T ds = double stranded DNA
ss = single stranded DNA E = Digested with EcoRI U = uncut
T = digested with TaqI T + T +
E U T E U T E U T
ds ss ds
On reviewing the known sequence (Chung et al, 1990), the EcoRI site lies upstream of the primer used and was therefore not in the PCR product, thus explaining why the samples did not digest with EcoRI. The results of the single stranded PCR can be explained if there are at least three Hindlll sites 6-700bp
and 1 .0 -1 .4kb apart at the 3' end of the a-fibrinogen gene and incomplete digestion of genomic DNA with Hindlll (figure 3.15). This is compatible with Kant's Hindllll restriction map of the fibrinogen gene cluster (Kant et al, 1985). For the production of double-stranded D N A using a single oligonucleotide there would have to be an identical or almost identical sequence to this oligonucleotide in the complementary strand of DNA where the Hindlll restriction sites are found. No explanation w as found why TaqI digested the known non-cutting allele and not the known cutting allele. In order to investigate these findings further, the NaR clone which contains the fibrinogen aR intergenic sequence w as subcloned and appropriate inserts sequenced.
3.3.1.b Subcloning of the fibrinogen aR intergenic region
T h e sam e bacteriophage
X
clone, NaR, was used and prepared as described in 3.2.2.a , up to the miniprep extraction of DNA and Hindlll digestion of the inserts which had been subcloned. Inserts of predicted length, 0.7kb and I.Okb (Kant et al, 1985 and own observations), were identified in several of the clones. Som e of the clones contained both inserts, which would support the findings of Kant et al, that the three most 5' Hindlll sites in the intergenic region lie within 1.7kb of each other. The individual inserts were double digested with Hindlll/TaqI and although the banding patterns were complex, they indicated that there w ere TaqI cutting sites in both the 0.7kb insert and the I.Okb inserts (not shown). Therefore the DN A sequence of selected 0.7kb and I.Okb inserts w as determined to enable amplification by P C R and subsequent digestion with TaqI. T h e orientation of the subcloned fragments is shown in figure 3.18.Figure 3.18
Intergenic region (not to sca le) o f the a and p fibrinogen genes and orientation o f su b clo n ed fragm ents
alph
F = forward primer R =reverse primer
Clones 1,4,6 and 16 indicated H = Hindlll restriction site
0.7kb I.Okb H H H I F 6R 1R 6F 4F 16F 4R 16R
beta
_________ Direction of transcription of fibrinogen genes ro3 .3 .1 .c D ire c t se q u e n c in g o f th e 0.7kb and I.O k b inserts
Tw o each of the 0.7kb and I.Okb inserts w ere partially sequenced initially using universal forward and reverse primers. New oligonucleotide primers were designed to allow further sequencing and/or PC R amplification of genomic DNA.
S e q u e n c e o f I.O k b in s e rt (su b clo n e s 4 and 16)
The sequence of the I.Okb insert using the fonA/ard primer is shown in figure 3.19 and the autoradiograph from the sequencing gel of the Alu consensus sequence with the TaqI site within it is shown in figure 3.20. The partial sequence of the 1 .Okb insert using the reverse primer is shown at appendix 3 and figure 3.21.
Fig u re 3.19 Sequence of I.Okb insert (subclones 4 and 16) fonA/ard primer The sequence of the pU C 18 vector is underlined with the oligonucleotide subsequently used for further sequencing shown in bold. W here it was impossible to identify whether a base was present or not, or repeated, these single bases are shown in bold. T h e TaqI site is shown in bold and underlined.
C G G G G A T C C T C TA G A G TC G A G C T G C A G C C C A A G C T T C T A G T T G C T C T T T A T T T A T G AAGGA A G A G A A A C A G C T A A C T C A G G G A T T G I I IT T AAAG T G AG TA GAG AT TGGGA GGATG G G G G A AAAAT A T G G G TG A G A TG A G A GATGA GGAGG TG G A G TA G G T G AG GT GAGAT T T G T G T T T A A
G A C G C T T G T A A T C C C A G C A C T T T G G G A G G C C G A G G G G G G T G AATC