Pengembangan pelacak DNA spesifik gen melalui bioinformatika: Indentifikasi gen penyandi protein biji 21 kDa pada kakao UAH Indonesia The development of gene-specific probe by bioinformatica: Identification of 21 kDa-encoding seed protein gene on Indonesian UAH cacao

SummaryA proper gene-specific probe is inevitablefor the process of gene discovery. In additionsuch probe can be utilized to study the expressionof corresponding gene. Probe, which is specificto gene of interest maybe developed usinginternet-accessible database coupled withmolecular techniques. This research aimed todevelop a probe, specific to 21 kDa-encodingseed protein gene and try it on cacao genomes.Two pairs of DNA primers were made based onthe conserved regions. Examined on the cacaogenomes with PCR technique demonstrated thatboth the specific and nested primer pairs wereable to amplify the targeted gene fragments withpredicted sizes, about 465 and 160 bp. RT-PCRwith total RNA from cacao seeds suggested thatthe specific primer can be utilized to determinethe expression level of the gene in the organ.Furthermore, Southern blotting analysis withgenomic DNA from five different cacao clones inIndonesia indicated that the probe wassignificantly specific to the gene. These concludethat a probe specific to the 21 kDa-encoding genewas developed and tested effective to identify thepresence and determine the expression of thegene.RingkasanProses penemuan gen memerlukan adanyapelacak spesifik gen tersebut. Selain itu, pelacakspesifik juga dapat digunakan dalam mempelajariekspresi suatu gen yang sesuai. Pelacak spesifikgen dapat dikembangkan dengan memanfaatkankemajuan bioinformatika teknik-teknik biologimolekuler. Penelitian ini bertujuan untukmerintis pengembangan pelacak spesifik gen danmengujinya pada genom kakao. Adapuntargetnya adalah gen penyandi protein 21 kDayang diekspresikan di biji kakao namun bukanmerupakan protein penyimpanan (storageprotein). Dua pasang primer DNA dihasilkan dariperancangan menggunakan dasar daerahterkonservasi. Pengujian di tingkat genom kakaodengan teknik PCR membuktikan bahwa keduapasangan primer tersebut dapat mengamplifikasisecara spesifik gen penyandi protein target. BaikPCR dengan pasangan primer spesifik genmaupun nested, terhadap dua klon kakao, masing-masing menghasilkan dua amplikon yangukurannya sesuai dengan ukuran prediksi, yaitusekitar 465 dan 160 pb-an. Pengujian dengan RT-PCR menunjukkan bahwa pelacak tersebut dapatdigunakan untuk menentukan ekspresi gentersebut dibiji kakao. Lebih dari itu, Southernblotting terhadap genom dari lima klon kakaoyang berbeda menegaskan bahwa pelacak gentersebut memiliki spesifisitas yang tinggi. Dengandemikian pelacak spesifik gen penyandi proteinbiji 21 kDa dapat dikembangkan dan terbuktiefektif untuk beberapa klon kakao di Indonesia.

Pengembangan pelacak DNA spesifik gen melalui bioinformatika: Indentifikasi gen penyandi protein biji 21 kDa pada kakao UAH Indonesia The development of gene-specific probe by bioinformatica: Identification of 21 kDa-encoding seed protein gene on Indonesian UAH cacao

SummaryA proper gene-specific probe is inevitablefor the process of gene discovery. In additionsuch probe can be utilized to study the expressionof corresponding gene. Probe, which is specificto gene of interest maybe developed usinginternet-accessible database coupled withmolecular techniques. This research aimed todevelop a probe, specific to 21 kDa-encodingseed protein gene and try it on cacao genomes.Two pairs of DNA primers were made based onthe conserved regions. Examined on the cacaogenomes with PCR technique demonstrated thatboth the specific and nested primer pairs wereable to amplify the targeted gene fragments withpredicted sizes, about 465 and 160 bp. RT-PCRwith total RNA from cacao seeds suggested thatthe specific primer can be utilized to determinethe expression level of the gene in the organ.Furthermore, Southern blotting analysis withgenomic DNA from five different cacao clones inIndonesia indicated that the probe wassignificantly specific to the gene. These concludethat a probe specific to the 21 kDa-encoding genewas developed and tested effective to identify thepresence and determine the expression of thegene.RingkasanProses penemuan gen memerlukan adanyapelacak spesifik gen tersebut. Selain itu, pelacakspesifik juga dapat digunakan dalam mempelajariekspresi suatu gen yang sesuai. Pelacak spesifikgen dapat dikembangkan dengan memanfaatkankemajuan bioinformatika teknik-teknik biologimolekuler. Penelitian ini bertujuan untukmerintis pengembangan pelacak spesifik gen danmengujinya pada genom kakao. Adapuntargetnya adalah gen penyandi protein 21 kDayang diekspresikan di biji kakao namun bukanmerupakan protein penyimpanan (storageprotein). Dua pasang primer DNA dihasilkan dariperancangan menggunakan dasar daerahterkonservasi. Pengujian di tingkat genom kakaodengan teknik PCR membuktikan bahwa keduapasangan primer tersebut dapat mengamplifikasisecara spesifik gen penyandi protein target. BaikPCR dengan pasangan primer spesifik genmaupun nested, terhadap dua klon kakao, masing-masing menghasilkan dua amplikon yangukurannya sesuai dengan ukuran prediksi, yaitusekitar 465 dan 160 pb-an. Pengujian dengan RT-PCR menunjukkan bahwa pelacak tersebut dapatdigunakan untuk menentukan ekspresi gentersebut dibiji kakao. Lebih dari itu, Southernblotting terhadap genom dari lima klon kakaoyang berbeda menegaskan bahwa pelacak gentersebut memiliki spesifisitas yang tinggi. Dengandemikian pelacak spesifik gen penyandi proteinbiji 21 kDa dapat dikembangkan dan terbuktiefektif untuk beberapa klon kakao di Indonesia.

Laboratorial diagnosis of fragile-X syndrome: experience in a sample of individuals with pervasive developmental disorders

Fragile X syndrome is a frequent genetic disease associated to developmental disorders, including learning disability, mental retardation, behavioral problems and pervasive developmental disorders (autism and related conditions). We studied a sample of 82 individuals (69 males and 13 females) presenting with pervasive developmental disorders using three techniques for the diagnosis of fragile X syndrome (FXS). Cytogenetic analysis detected the fragile site in four males, but only one showed a consistent positive rate. Molecular study based on the PCR technique was inconclusive for most females (92.3%), which where latter submitted to Southern blotting analysis, and for one male (1.4%), excluding the FRAXA mutation in the remaining male individuals (98.6%). Molecular tests using the Southern blotting technique confirmed only one positive case (1.2%) in a male subject. These results showed that Southern blotting analysis of the FRAXA mutation has the best sensitivity and specificity for the diagnosis of FXS but also validated the PCR technique as a confinable screening test.

RAR-alpha gene rearrangements as a genetic marker for diagnosis and monitoring in acute promyelocytic leukemia

Acute promyelocytic leukemias (APLs) are characterized by a translocation that involves chromosomes 15 and 17. The translocation breakpoints have recently been identified and shown to involve the RAR- alpha gene on 17 and myl on 15. Here we report Southern blotting analysis of 26 APLs, including cases with normal karyotypes and atypical morphology, which showed RAR-alpha rearrangements in 92% cases, myl rearrangements in 73%, and either RAR-alpha or myl rearrangements in 100%. Despite a negative clinical and morphologic picture, DNA rearrangement analysis showed that neoplastic promyelocytes persisted in the bone marrow of two patients sampled after induction chemotherapy. Therefore, the RAR-alpha and myl rearrangements provide molecular markers for accurately diagnosing APLs and monitoring the course of the disease during and after chemotherapy.

RAR-alpha gene rearrangements as a genetic marker for diagnosis and monitoring in acute promyelocytic leukemia

Acute promyelocytic leukemias (APLs) are characterized by a translocation that involves chromosomes 15 and 17. The translocation breakpoints have recently been identified and shown to involve the RAR- alpha gene on 17 and myl on 15. Here we report Southern blotting analysis of 26 APLs, including cases with normal karyotypes and atypical morphology, which showed RAR-alpha rearrangements in 92% cases, myl rearrangements in 73%, and either RAR-alpha or myl rearrangements in 100%. Despite a negative clinical and morphologic picture, DNA rearrangement analysis showed that neoplastic promyelocytes persisted in the bone marrow of two patients sampled after induction chemotherapy. Therefore, the RAR-alpha and myl rearrangements provide molecular markers for accurately diagnosing APLs and monitoring the course of the disease during and after chemotherapy.