DNA fragmentation is a hallmark of apoptosis that is induced by apopto-tic stimuli in various cell types. Apoptotic signal pathways, which eventu-ally cause DNA fragmentation, are largely mediated by the family of
cysteinyl aspartate-specific protease caspases.
The main contents of this chapter include all of the following: Sequence-specific DNA fragmentation, cloning fragments of DNA, hybridization, the polymerase chain reaction, DNA sequence analysis, bioinformatics: information technology and genomes, the hemoglobin genes: a comprehensive example.
DNA fragmentation is a hallmark of apoptosis that occurs in a variety of
cell types; however, it remains unclear whether caspase-3 is required for its
induction. To investigate this, we produced caspase-3 knockout Chinese
hamster ovary (CHO)-K1 cells and examined the effects of gene knockout
and treatment with caspase-3 inhibitors.
Kỹ thuật RFLP là kỹ thuật nghiên cứu tính đa hình chiều dài của các phân đoạn DNA dựa trên điểm cắt các enzim giới hạn. Khi ủ DNA với enzim giới hạn ở dung dịch đệm thích hợp ở pH, nhiệt độ thích hợp sẽ sẽ tạo ra những phân đoạn DNA với kích thước khác nhau, từ đó lập nên các bản đồ gen. Tham khảo chương hai "Kỹ thuật RFLP - Restriction fragment length polymorphisms" để hiểu hơn về vấn đề này.
The Hitachi SV1100 utilizes capillary electrophoresis on a
microchip that is capable of rapidly sizing DNAfrag-ments. Reproducibility of electrophoresis in different
channels was shown by comparing the migration times of
the internal controls, DNAfragments of 100 and 800 bp.
The range of DNAsizing for this microchip is between
100 and 800 bp, and accuracy in sizing of a 322 bp DNA
fragment of a pUC118 PvuII digest was observed, inde-pendent of DNAconcentration. Although relatively good
quantification of this fragment was observed with a DNA
concentration of 1.
We report on a sequence-specific double-stranded DNA labelling strategy
in which a stem–loop triplex forming oligonucleotide (TFO) is able to
encircle its DNA target. Ligation of this TFO to either a short hairpin
oligonucleotide or a long double-stranded DNA fragment leads to the for-mation of a topological complex. This process requires the hybridization of
both extremities of the TFO to each other on a few base pairs. The effects
of different factors on the formation of these complexes have been investi-gated. ...
After studying this chapter you will be able to understand: DNA cloning permits production of multiple copies of a specific gene or other DNA segment, restriction fragment analysis detects DNA differences that affect restriction sites, entire genomes can be mapped at the DNA level, genome sequences provide clues to important biological questions, the practical applications of DNA technology affect our lives in many ways.
Kỹ thuật AFLP (Amplified Fragments Length Polymorphism) được hiểu là sự đa
dạng của các đoạn DNA được nhân lên có định hướng sau khi bị cắt bởi 2 enzim
giới hạn, sử dụng những phân đoạn DNA làm khuôn cho phản ứng khuếch đại
PCR. AFLP là một trong những kỹ thuật in dấu DNA được phát triển bởi Vos và
cộng sự năm 1995.
Kỹ thuật RFLP (viết tắt từ Restriction Fragment Length Polymorphism) hay kỹ thuật Đa hình chiều dài đoạn cắt giới hạn) là kỹ thuật nghiên cứu tính đa hình DNA bằng cách kết hợp kỹ thuật PCRvà phản ứng cắt của enzyme giới hạn (Restriction Enzyme, RE).
This book contains four parts with 16 chapters. Firstly, an optimal stimulation scheme
for ovaries, particularly natural and minimal stimulation of ovaries, has been
discussed in the first part. Then, one paper analyzed that how many oocytes per
retrieval will be the best for human IVF practice. If one stimulation scheme produces
too many eggs, it often results in hyperstimulation syndrome.
Nucleotide Repeat Expansion Disorders
Several diseases are associated with an increase in the number of nucleotide repeats above a certain threshold (Table 62-6). The repeats are sometimes located within the coding region of the genes, as in Huntington disease or the X-linked form of spinal and bulbar muscular atrophy (SBMA, Kennedy syndrome). In other instances, the repeats probably alter gene regulatory sequences. If an expansion is present, the DNA fragment is unstable and tends to expand further during cell division.
Integration of cell death responses. Cell death through an apoptotic mechanism requires active participation of the cell. In response to interruption of growth factor (GF) or propagation of certain cytokine death signals (e.g., tumor necrosis factor receptor, TNF-R), there is activation of "upstream" cysteine aspartyl proteases (caspases), which then directly digest cytoplasmic and nuclear proteins, resulting in activation of "downstream" caspases; these cause activation of nucleases, resulting in the characteristic DNA fragmentation that is a hallmark of apoptosis. ...
Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated
transcription factor with two heme-binding sites on two PAS domains. In
the present study, we compared the optical absorption spectra, resonance
Raman spectra, heme-binding kinetics and DNA-binding characteristics of
the isolated fragment containing the N-terminal basic helix–loop–helix
(bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the
PAS-A domain alone.
More discrete sequence alterations rely heavily on the use of the PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. Screening for point mutations can be performed by numerous methods (Table 62-9); most are based on the recognition of mismatches between nucleic acid duplexes, electrophoretic separation of single- or double-stranded DNA, or sequencing of DNA fragments amplified by PCR.
RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Delivering sequences of RNAi in vivo remains a problem. The aim of this study was to use JC virus (JCV) virus-like particles (VLPs) as a vector for delivering RNAi in silencing the cytokine gene of IL-10. Methods: JCV VLPs were generated by recombinant JCV VP1 protein in yeast expression system. DNA fragment containing IL-10 shRNA was packaged into VLPs by osmotic shock. Results: In RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone.
Sequence analysis of aPaenibacillussp. BP-23 recombinant
clone coding for a previously described endoglucanase
revealed the presence of an additional truncated ORF with
homology to family 48 glycosyl hydrolases. The corres-ponding 3509-bp DNA fragment was isolated after gene
walking and cloned inEscherichia coliXl1-Blue for expres-sion and purification. The encoded enzyme, a cellulase
of 1091 amino acids with a deduced molecular mass of
118 kDa and a pI of 4.
Cell death-inducing DFF45-like effector (CIDE) family proteins, including
cell death-inducing DFF45-like effector A (CIDEA), cell death-inducing
DFF45-like effector B (CIDEB) and cell death-inducing DFF45-like effec-tor C (CIDEC) [fat-specific protein of 27 kDa in rodent (FSP27) in
rodents], were originally identified by their sequence homology to the
N-terminal region of DNA fragmentation factor DFF40⁄45.
Restriction landmark genomic scanning (RLGS) is a powerful method for
the systematic detection of genetic mutations in DNA length and epigenetic
alteration due to DNA methylation. However, the identification of poly-morphic spots is difficult because the resulting RLGS spots contain very
little target DNA and many non-labeled DNA fragments.