How can we achieve cloning of mRNA's complete sequence?

In RACE technique, PCR is used to amplify partial region between a single point in a mRNA transcript and its 3'- or 5'-end. The premise is that a short internal stretch of the sequence from the target mRNA is already known. Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved by using primers that anneal to the preexisting poly(A) tail (3'-end) or to an appended homopolymer tail (5'-end). Using RACE, enrichments on the order of 106- to 107-fold can be obtained. To generate "5'-end" partial cDNA clones, reverse transcription (primer extension) should be carried out by using a gene-specific primer to generate first-strand products. Then, a Poly(A) tail is appended using terminal deoxynucleotidyltransferase (TdT) and dATP. Amplification is then achieved to form the second strand of cDNA. Finally, a second set of PCR cycles is carried out using nested primers to increase specificity.

According to statistics in the past, most attempts to identify and isolate a novel cDNA will result in clones that represent only a part of the mRNA's complete sequence. Then how could we achieve cloning of mRNA's complete sequence?

The answer should be PCR in RACE technique. The missing sequence (cDNA ends) can be cloned by PCR, using the technique called rapid amplification of cDNA ends (RACE).

RACE cloning has several advantages for mRNA target cloning:

 It takes only weeks to screen cDNA libraries,
 Can obtain individual cDNA clones
 Can analyze the clones to determine if the missing sequence is present and such information can be generated within a few days.                          

Because of those advantages, RACE cloning has becomes popular in the application to modify RNA preparation and/or reverse transcription conditions until full-length cDNAs are generated and observed.

The principle of RACE technique


Creative Biogene’s RACE kits

Creative Biogene’s RACE kits are specially designed for the amplification of DNA sequences from a messenger RNA template between a defined internal site and unknown sequences of either the 3' or the 5' end of the mRNA. The 5' RACE kits contain all necessary components for performing 5' RACE. The kits can achieve the amplification of rare messages with little sequence information. And the PCR products can be used for sequencing and cloning.

More information at http://www.creative-biogene.com/5-RACE-RTRZ-02-1232898-71.html

WHO Targets the Indoor Air Pollution

WHO Targets the Indoor Air Pollution 

The World Health Organization (WHO) has started to tackle on the indoor pollution problems and this Wednesday it launched its first guidelines for improving indoor air quality and set targets for reducing emissions of domestic health-damaging pollutants.

The guidelines for indoor air quality were released right after its previous report on indoor air pollution. The guidelines highlight the importance to use cleaner home energy sources especially for people of developing countries.

“Ensuring cleaner air in and around the home is fundamental to reducing the burden of disease from air pollution, especially in low-and middle-income countries,” says Dr Maria Neira, WHO Director for Public Health, Environmental and Social Determinants of Health.

WHO’s earlier report this year revealed that about 4.3 million people die every year from household air pollution emitted by cook stoves, space heaters and fuel-based lamps. And there are about 3 billion people worldwide still inaccessible to clean fuels.

Scientists warned that the high levels of fine particulate matter and carbon monoxide, released by the burning of solid fuels in inefficient stoves, space heaters or lamps would increase the risks of stroke, ischemic heart disease and chronic obstructive pulmonary disease.

The guidelines set the emissions targets for different kinds of domestic appliances and those standards are established after years of review and examination of the health impacts of household air pollution emissions.

The new guidelines also call on people to stop using unprocessed coal as household fuel for the reason that incomplete combustion of coal in inefficient stoves and space heaters can lead to severe illness and premature death. And the use of kerosene as a household fuel is also discouraged for its adverse impact on air quality and safety and the possibility to cause burns, fires and poisoning.

According to WHO, women and young children should particularly be aware of this indoor air pollution because they spend the most time at home taking in the pollutant air. More than 50% of pneumonia deaths among children under 5 are linked to household air pollution, according to WHO.

In order to meet the new targets, cleaner and more modern cooking and heating appliances are needed. People, especially those who live in low-and-middle income regions should no longer ignore the indoor air quality for the sake of our health and safety. And also governments, non-governmental organizations, the private sector and development partners should work together to help with the implementation of these guidelines.

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Bioscience Industry Boosts Buffalo Niagara’s Economy

A recent economic-impact analysis report by an industry trade group shows that employment in the biosciences sector is growing at a faster speed in Western New York than in Upstate New York and the state as a whole, fueled by strong performance locally in the areas of biomedical research, medical testing and drug distribution.

The biosciences sector is a focus of economic-stimulus efforts in Buffalo Niagara and in Albany. The state has invested tens of millions of dollars to boost drug development and biomedical research here. According the report, the industry provides jobs for more than 7,400 people in the region, up 4 percent over the most recent five-year period, producing a $5.6 billion total economic impact.

Bioscience jobs are valued here because these jobs pay more than jobs in other industries. The report shows average biomedical job in Upstate New York pays nearly $71,900, roughly $30,000 more than the $41,373 average private-sector job.

The report also shows that every bioscience job in Western New York supports an additional 1.7 jobs at vendor or supply-chain companies and 21,768 workers directly or indirectly are tied to the industry here, earning $1.3 billion money in total.

This industry is valued not only for its great contribution to the local employement, but also its ability to pull in billions of dollars in research and development funding to this region every year.

Thought the industry develops well those years, bioscience companies have to reinvent themselves in the face of new regulations featured in the Affordable Care Act, including a fundamental reshaping of the health-care payment model and a tax on medical-device makers and other changes.

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The Application of Expression Vector

An expression vector, also known as an expression construct, is usually a plasmid or virus artificially made for protein expression in cells. The working principle of expression vector is to introduce a specific gene into a target cell, thus controlling the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. Expression vectors are recognized as basic tools for biotechnology and the production of proteins.

1.       Application in protein production

Expression vector in an expression host is the usual method used in laboratories to produce proteins for research. Most protein pharmaceuticals are now produced through recombinant DNA technology using expression vectors. A well-designed expression vector will produce a significant amount of stable messenger RNAs and proteins. These peptide and protein pharmaceuticals may be hormones, vaccines, antibiotics, antibodies, and enzymes.

2.       Application in transgenic products

In recent years, expression vectors have been used to introduce specific genes in organisms, for example in agriculture it is used to produce transgenic plants. Expression vectors have been used to introduce a vitamin A precursor, beta-carotene, into rice plants. They are used to extend the ripeness of tomatoes by altering the plant so that it produces less of the chemical that causes the tomatoes to rot.

3.       Application in gene therapy

Expression vectors are also being applied in gene therapy. Gene therapy is a promising treatment for a number of diseases where a "normal" gene carried by the vector is inserted into the genome, to replace an "abnormal" gene or supplement the expression of particular gene. Although this treatment is still a risky option due to the viral vector used which can cause ill-effects, it is still being used for its promising results.

About us

Creative Biogene is a US-based manufacturer and provider of genomics and proteomics products and services for academic and governmental research institutes, pharmaceutical and biotechnology industry. It provides products such as kits, reagents, vectors and services that help researchers explore questions about gene discovery, regulation, and function. Creative Biogene offers a wide range of vectors including expression vectors and clone vectors and etc.

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Gene Once Beneficial to Human May Also Become Harmful to Health

Source from: Science Daily

A recent research reported that a genetic variant of ancient gene discovered in individuals living in the Arctic , which most likely provided an evolutionary advantage for processing high-fat diets or for surviving in a cold environment in past, however also seems to increase the risk of hypoglycemia, or low blood sugar, and infant mortality in today's northern populations.

The finding was published online at October 23 in Cell Press's American Journal of Human Genetics. It serves as an example to show us that how an initially beneficial genetic change could be detrimental to future generations.

A senior author Dr. Toomas Kivisild from the University of Cambridge said that their work discovered a case in which the same variant once had likely been selectively advantageous in the past could become disadvantageous under current environmental conditions.

Dr. Kivisild and his colleagues firstly analyzed the genomes collected from 25 individuals from Northern Siberia. Then they compared these gene sequences of arctic people with those from 25 people from Europe and 11 from East Asia.

The team successfully identified a variant of gene that was unique to Northern Siberians and was located within CPT1A, a gene that encodes an enzyme involved in the digestion of long fatty acids, which are prevalent in meat-based diets.

Agriculture is unsustainable in Arctic regions because of the extremely cold environment. Therefore coastal populations there have historically fed mostly on marine mammals. They believed this variant of gene must have something to do with those arctic people’s digest system.

However, their later survey showed that the high frequency of this variant in these populations had been proved to be harmful to people’s health. When the investigators looked at the global distribution of the CPT1A variant, they found that it was present in 68% of individuals in the Northern Siberian population and had not been found in other publicly available genomes. Surprisingly, they also found that the variant had previously been linked to high infant mortality and hypoglycemia in Canadian Inuits.

This study results show people the importance to see things from the perspective of development and for scientists, they also need to have an evolutionary understanding of past and then to better explain the present.

About us

Creative Biogene is a US-based manufacturer and provider of genomics and proteomics products and services for academic and governmental research institutes, pharmaceutical and biotechnology industry. It provides products such as kits, reagents, enzymes and sevices like cloning service, RNAi services.

Biological Functions of RNA Interference

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific messenger RNA (mRNA) molecules. Andrew Fire, an American biologist and professor of pathology and of genetics at the Stanford University School of Medicine and Craig C. Mello, another American biologist and professor of molecular medicine at the University of Massachusetts Medical School in Massachusetts firstly discovered RNA Interference. They published their work on RNAi in 1998, which won them the 2006 Nobel Prize in Physiology or Medicine.

There are two types of small ribonucleic acid (RNA) molecules: microRNA (miRNA) and small interfering RNA (siRNA). Those two are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific mRNA molecules and to increase or decrease their activity.

RNAi is a valuable research tool, both in cell culture and in living organisms, because synthetic double-stranded RNA (dsRNA) introduced into cells can selectively and robustly induce suppression of specific genes of interest. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long dsRNA molecules into short double stranded fragments of ~20 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs, respectively the passenger strand and the guide strand.

There are three major biological functions of RNA interference:

1. Immunity

RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may also prevent the self-propagation of transposons.

2. Downregulation of genes

Endogenously expressed miRNAs, including both intronic and intergenic miRNAs, are most important in translational repression and in the regulation of development. miRNA activity is particularly wide-ranging and regulates entire gene networks during development by modulating the expression of key regulatory genes.

3. Upregulation of genes

RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon dubbed RNA activation.

About us

Creative Biogene is one of the world’s leading suppliers of biotechnological and biochemical products and services. We provide leading functional genomics technologies including RNAi vector and RNAi service for gene silencing and cutting-edge systems for gene editing and gene knockout. All RNAi research projects carried out by Creative Biogene are custom-designed to address each client's specific experimental, strategic and budgetary guidelines, while always meeting the highest scientific standards in the field.

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