Molecular Genetic Diagnosis of Human Malignant Hyperthermia
S. Tsuyoshi Ohnishi, Tomoko Ohnishi in Malignant Hyperthermia, 1994
Once linkage to a polymorphic marker is established, genetic diagnosis is feasible. However, optimal diagnosis and a complete understanding of the underlying pathophysiology relies on full characterization of the mutant allele(s) of the disease gene. Tightly linked markers may still be 1 to 5 cM from the disease locus, and several million nucleotides containing a few or many genes must be analyzed. Using the marker as a starting point, chromosome walking is begun by the isolation of a clone from a library of DNA fragments, which is known in advance to contain the marker. The goal is to build up a series of overlapping clones in phage, cosmid, or yeast libraries in order to define sequence further and further from the RFLP starting point, and closer to the disease gene. Smaller fragments of the cloned segment are then mapped and oriented so that overlapping regions are identified, and the clones aligned to reflect the original order of the fragments along the chromosome. By restriction enzyme mapping or direct sequencing, each new clone can be compared to the original clone and used to search the library for additional clones. Regions rich in repetitive DNA may be extremely difficult to clone, and methods are needed to cover the great length of DNA that may span a marker locus to disease gene. In chromosome jumping, rather than cloning and characterizing every fragment between the marker and disease loci, uninteresting regions of DNA are skipped or jumped by circularizing large DNA fragments with a selectable marker and bringing the two ends together in a loop. The loop is then ligated and the fragments containing the linked ends are cloned for screening with specific probes. With the ability to clone up to 1 Mb (106 nucleotides) of human DNA in yeast, chromosome walking with yeast libraries accomplishes the same objective as chromosome jumping, but with a substantial reduction in effort and time.
Molecular Approaches Towards the Isolation of Pediatric Cancer Predisposition Genes
John T. Kemshead in Pediatric Tumors: Immunological and Molecular Markers, 2020
Histopathology of WT is variable, but is normally characterized by undifferentiated blastemal cells similar to those seen in the developing kidney. Grobstein105 showed that differentiation of the kidney mesenchyme was triggered by a factor produced by the ureter bud. Possibly, disruption of this induction system could allow continuous proliferation of blastemal cells which overcome the growth limitations imposed by differentiation. The implication that specific genes in region 11p13 are involved with the predisposition to WT and other rare embryonic tumors has prompted several groups to look for genes in 11p13 showing abnormal expression of the type described for retinoblastoma. Several approaches being used will be discussed below, but all depend on the isolation of DNA sequences from within the 11p13 region. One option would be to use existing 11p13 markers to attempt chromosome walking towards the Wilms’ locus. Chromosome walking is the sequential isolation of clones which are normally adjacent within the genome (Figure 13). Walking is relatively time consuming and requires the isolation of the end regions of each clone in order to achieve the maximum step. These procedures can be carried out in either lambda or cosmid vectors. The isolation of a single overlapping clone constitutes a “step” and a series of steps constitutes a “walk”. Such strategies for walking long distances in the human genome are not practical for a variety of reasons.106 Cosmid walking offers the best opportunity for covering large distances and the development of new vectors such as the one described by Little and Cross107 might make walking slightly quicker. Into their cosmid vector, LORIC,107 the SP6 and T7 promoters have recently been introduced.108 This allows rapid generation of radioactive RNA probes directly from the end regions of the clone which can be used in the next walking step (see Figure 13).
A Survey of Newer Gene Probing Techniques
Victor A. Bernstam in Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
The top-down approach identifies and makes assignments of progressively smaller fragments of DNA from a given chromosome. The bottom-up strategy identifies and aligns, in a continuum of sequences, overlapping sets of DNA clones (contigs). Techniques known as chromosome walking, chromosome jumping, and probe walking are used in these strategies.
Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51
Published in Biofouling, 2018
Xiaochun Huang, Chang Chen, Chunhua Ren, Yingying Li, Yiqin Deng, Yiying Yang, Xiongqi Ding
Figure 1To find the possible genetic differences between the ZJ-T and ZJ-O strains, their genetic profileswere compared by PFGE. Four different restriction enzymes were used to digest the bacterial chromosomes, and only digestion of NotI revealed different band profiles between the two variants. As shown in Figure 1, there is only one band difference, where a larger band is present in ZJ-T while a smaller one is present in ZJ-O. The DNA contained in the larger band was extracted from the gel of ZJ-T strain and sequenced using the primer walking method (Tail-PCR).
Molecular spectrum of Hb H disease and characterization of rare deletional α-thalassemia found in Thailand
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2020
Wittaya Jomoui, Wanicha Tepakhan, Surada Satthakarn, Sitthichai Panyasai
MLPA analysis could predict deletional α-thalassemia in four patients (P1-P4) in this study. Patient P1 was found to have a large deletional α0-thalassemia confirmed as the (–SA deletion). The (–SA deletion) was originally found in a patient from South Africa which removed both α2 and α1 genes, causing α0-thalassemia [21]. This (–SA deletion) has a deletion of approximately 23 kb with an insertion of 157 bases [18]. In Thailand, the (–SA deletion) has not been documented. In this study, we first report rare Hb H disease related to a large deletion (–SA deletion) with α+-thalassemia (-α3.7) in the Thai population. Patient P2 was found to have a large novel deletional α0-thalassemia (approximately 31-62 kb) in this region, which the MLPA analysis showed was unrelated to four large deletions (–SEA deletion, –THAI deletion, –FIL deletion, –MED deletion) previously described in Southeast Asia [22]. Unfortunately, we could not characterize the breakpoint in this case by primer walking. Patient P3 was confirmed as a rare Hb H genotype related to rare α+-thalassemia (-α16.6 deletion) with α0-thalassemia (–SEA deletion). The large -α16.6 kb deletion removed the ψζ1-, ψζ2 -, ψα1- and α2-globin genes and remaining α1-globin genes. The -α16.6 deletion allele has been documented in Thailand; however, this deletion is rarely found with α+-thalassemia in this region [19,23]. Molecular detection of these rare deletion types should be implemented in a routine setting, especially α0-thalassemia as it can cause Hb Bart’s hydrops fetalis syndrome.
A genomic sequence of the type II-A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system in Mycoplasma salivarium strain ATCC 29803
Published in Journal of Oral Microbiology, 2022
Harumi Mizuki, Yu Shimoyama, Taichi Ishikawa, Minoru Sasaki
In ATCC 29803, the sequence of the PCR amplicon generated using the CAS9 FW1 and CAS9 RV2 primer pair was determined. The complete cas9 gene sequence was then obtained via assembly of this sequence and the flanking sequence of the CRISPR array. A region of the cas9 gene sequence was also analyzed via PCR amplification using the primer pair CAS9 FW3 and CAS9 RV3 (Table 2), as shown in Figure 1, and capillary sequencing with paired-end reading. The analyzed sequence (480 bp) completely matched the sequence obtained via primer walking (sequence data are not shown).
Related Knowledge Centers
- Chemical Synthesis
- Polymerase Chain Reaction
- Restriction Fragment Length Polymorphism
- Sanger Sequencing
- Shotgun Sequencing
- Plasmid
- Primer
- Chromosome Jumping
- Chromosome Landing