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Terminators

small plasmid vectors

it stands for the protein’s molecular weight of 90 kDa

Serial Analysis of Gene Expression

~1.8 Mb

~ 30%

PacBio

1. RNA interference (RNAi)

2. Antisense oligonucleotides (ASOs)

3. CRISPR/Cas9-mediated gene editing

Nanopore sequencing

1. Affinity Chromatography (as Co-immunoprecipitation)

2. Yeast two-hybrid (Y2H) system

   
   

3. [Bioluminescence resonance energy transfer (BRET)]

4. [Fluorescence resonance energy transfer (FRET)]

Synthetic lethal screening is a method used to identify genes that are essential for cell survival in the presence of a specific genetic or environmental perturbation. The goal of synthetic lethal screening is to identify pairs of genes where the loss of function of one gene is tolerated by the cell, but the simultaneous loss of function of both genes results in cell death.

There are several methods that can be used for synthetic lethal screening, including:

1. RNA interference (RNAi) screening

2. CRISPR-Cas9 screening:

3. High-throughput screening (HTS) using small molecule libraries

4. Combinatorial screening

Frederick Sanger

13.986

1. Template preparation:

   • original: the DNA template was isolated and purified from the sample and then used to create multiple copies of the DNA fragment using bacterial cloning vectors

   • modern: the DNA template is usually amplified using PCR, which is faster and more efficient than bacterial cloning.

2. Dye terminator chemistry:

   • original: used four separate reactions, each with a different labeled ddNTP, to terminate DNA synthesis at each nucleotide position.

   • modern: uses a single reaction that contains all four labeled ddNTPs, each with a different fluorescent dye, which allows for simultaneous detection of all four bases.

3. Detection and analysis:

   • original: the terminated DNA fragments were separated by gel electrophoresis, and the bands were visualized using autoradiography.

   • modern: the terminated fragments are separated by capillary electrophoresis, and the fluorescent signals are detected by a laser scanner and analyzed by a computer.

The use of a single reaction containing all four fluorescently labeled ddNTPs is one of the main reasons why the modern Sanger sequencing method is faster and more efficient than the original method. This approach eliminates the need for multiple reactions and simplifies the sample preparation process, making it possible to sequence multiple DNA fragments in a single run.

Synthetic lethality is a phenomenon in which the combination of two mutations or treatments, each of which alone is viable or even benign, results in cell death or impairment.

(Specifically, in synthetic lethal interactions, the simultaneous perturbation of two genes or pathways results in cell death, while the perturbation of either gene alone does not. The concept of synthetic lethality has important implications in cancer biology, as it provides a potential strategy for developing targeted therapies that selectively kill cancer cells with specific genetic mutations or alterations, while sparing normal cells. For example, if a cancer cell has a mutation in one gene, a synthetic lethal drug could be designed to target another gene in the same pathway, causing cell death only in the cancer cells, but not in normal cells.)

both next-generation sequencing (NGS) platforms that use different technologies to generate sequence data.

1. Sequencing technology:

   • Ion Torrent: uses semiconductor-based sequencing

   • 454 : uses pyrosequencing.

1. Read length:

   • Ion Torrent :typically generates shorter reads (up to ~400 base pairs),

   • 454: generates longer reads (up to ~1,000 base pairs).

2. Throughput:

   • Ion Torrent: generally has higher throughput than 454, allowing for more reads to be generated in a shorter amount of time.

3. Error rates:

   • Ion Torrent has higher error rates than 454, particularly in homopolymeric regions, which can affect the accuracy of the generated sequences.

4. Cost:

   • Ion Torrent is generally less expensive than 454, making it more accessible to researchers with smaller budgets.

N50 contig length is a statistic used in genome assembly to describe the length of the shortest contig that covers 50% of the total length of the assembled genome. It is a measure of the assembly quality and is useful for comparing different assemblies or different sequencing technologies.

Heat-shock proteins (HSPs) are a family of proteins that are produced by cells in response to stress, such as exposure to high temperatures, toxins, or other environmental factors. They are called heat-shock proteins because they were first discovered in cells that had been exposed to high temperatures, which causes the proteins to become denatured and lose their normal shape.

In addition to their role in stress response, HSPs have been implicated in a wide range of cellular processes, including protein synthesis, transport, and degradation, and in the regulation of cell cycle and apoptosis. Dysregulation of HSPs has been linked to a number of diseases, including cancer, neurodegenerative disorders, and inflammatory diseases, making them a subject of ongoing research and therapeutic development.

1. Definition:

   • Faux reads: are artificially created reads that simulate sequencing data

   • shotgun reads: are randomly generated reads from fragmented DNA.

2. Purpose:

   • Faux reads: are used to evaluate the accuracy and performance of bioinformatics tools

   • shotgun reads: are used to sequence and assemble entire genomes.

3. Source of data:

   • Faux reads: are generated by simulating sequencing data based on a known reference genome

   • shotgun reads: are obtained from the actual DNA sample being sequenced.

4. Coverage:

   • Faux reads: are typically generated at high coverage to assess the accuracy and sensitivity of bioinformatics tools

   • shotgun reads: are generated at variable coverage depending on the sequencing technology and research question.

5. Read length:

   • Faux reads: can be generated at various read lengths, depending on the research question

   • shotgun reads: are typically shorter due to the limitations of current sequencing technologies.

6. Assembly:

   • Faux reads: are not typically used for genome assembly

   • shotgun reads: are the primary data used for genome assembly.

In genome assembly, contigs and scaffolds are two different types of sequences that are assembled from the overlapping reads obtained from the sequencing data.

A contig is a continuous sequence of DNA that is assembled from the overlapping reads obtained from the sequencing data. Contigs are usually generated by de novo assembly algorithms and represent a set of overlapping reads that form a contiguous sequence with no gaps. However, contigs may not represent the complete genome sequence, as they may have gaps or misassemblies due to repetitive regions in the genome or errors in the sequencing data.

A scaffold, on the other hand, is a set of contigs that are ordered and oriented relative to each other based on additional information, such as mate-pair information or optical mapping data. A scaffold can fill gaps and resolve ambiguities in the assembly to provide a more complete and accurate representation of the genome. A scaffold may also contain gaps, which are regions of the genome where the sequence is unknown.

In summary:

contig: is a contiguous sequence of DNA assembled from overlapping reads

scaffold: is a set of contigs that are ordered and oriented relative to each other to provide a more complete and accurate representation of the genome

Pyrosequencing is a DNA sequencing technology that was used by the 454 Life Sciences sequencing platform, which is now discontinued. Therefore, pyrosequencing was mainly used by the 454 sequencing method. However, other sequencing platforms such as Ion Torrent and Roche GS FLX also use similar sequencing-by-synthesis technologies.

The PacBio sequencing method relies on real-time sequencing, also known as single-molecule, real-time (SMRT) sequencing. This technology utilizes a DNA polymerase enzyme attached to a DNA template, and the incorporation of fluorescent nucleotides generates a real-time signal that can be detected and recorded. This approach allows for longer read lengths compared to other sequencing technologies, and has applications in de novo genome assembly, transcriptome analysis, and epigenetic studies.