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How long can I store extracted RNA?


If I extract RNA from a (leaf tissue) sample using a one-step phenol:chloroform extraction, how long can those samples be stored at -80°C? And how many times can I defrost and refreeze them before they will become degraded?


I've found that extracted RNA using commercial kits has stayed stable for many years at -80 C. I would certainly aliquot it before freezing however as RNA is particularly sensitive to freeze-thaw cleavage.


We can keep extracted RNA in -80°C for a few weeks, but before the start of any experiments, it needs to be validated by gel electrophoresis.


I keep my RNA in 1mM sodium citrate pH 6.4. Citrate is a chelator, and helps trap the divalent metals many RNAses need to work. The lower pH also helps inhibit RNAse activity. EDTA could work as a chelator, but it only chelates well at pH's where RNAse activity is worse. Citrate gives both chelation and low pH.

Of course I also keep my RNA at -80.


Biologists 'transfer' a memory through RNA injection

UCLA biologists report they have transferred a memory from one marine snail to another, creating an artificial memory, by injecting RNA from one to another. This research could lead to new ways to lessen the trauma of painful memories with RNA and to restore lost memories.

"I think in the not-too-distant future, we could potentially use RNA to ameliorate the effects of Alzheimer's disease or post-traumatic stress disorder," said David Glanzman, senior author of the study and a UCLA professor of integrative biology and physiology and of neurobiology. The team's research is published May 14 in eNeuro, the online journal of the Society for Neuroscience.

RNA, or ribonucleic acid, has been widely known as a cellular messenger that makes proteins and carries out DNA's instructions to other parts of the cell. It is now understood to have other important functions besides protein coding, including regulation of a variety of cellular processes involved in development and disease.

The researchers gave mild electric shocks to the tails of a species of marine snail called Aplysia. The snails received five tail shocks, one every 20 minutes, and then five more 24 hours later. The shocks enhance the snail's defensive withdrawal reflex, a response it displays for protection from potential harm. When the researchers subsequently tapped the snails, they found those that had been given the shocks displayed a defensive contraction that lasted an average of 50 seconds, a simple type of learning known as "sensitization." Those that had not been given the shocks contracted for only about one second.

The life scientists extracted RNA from the nervous systems of marine snails that received the tail shocks the day after the second series of shocks, and also from marine snails that did not receive any shocks. Then the RNA from the first (sensitized) group was injected into seven marine snails that had not received any shocks, and the RNA from the second group was injected into a control group of seven other snails that also had not received any shocks.

Remarkably, the scientists found that the seven that received the RNA from snails that were given the shocks behaved as if they themselves had received the tail shocks: They displayed a defensive contraction that lasted an average of about 40 seconds.

"It's as though we transferred the memory," said Glanzman, who is also a member of UCLA's Brain Research Institute.

As expected, the control group of snails did not display the lengthy contraction.

Next, the researchers added RNA to Petri dishes containing neurons extracted from different snails that did not receive shocks. Some dishes had RNA from marine snails that had been given electric tail shocks, and some dishes contained RNA from snails that had not been given shocks. Some of the dishes contained sensory neurons, and others contained motor neurons, which in the snail are responsible for the reflex.

When a marine snail is given electric tail shocks, its sensory neurons become more excitable. Interestingly, the researchers discovered, adding RNA from the snails that had been given shocks also produced increased excitability in sensory neurons in a Petri dish it did not do so in motor neurons. Adding RNA from a marine snail that was not given the tail shocks did not produce this increased excitability in sensory neurons.

In the field of neuroscience, it has long been thought that memories are stored in synapses. (Each neuron has several thousand synapses.) Glanzman holds a different view, believing that memories are stored in the nucleus of neurons.

"If memories were stored at synapses, there is no way our experiment would have worked," said Glanzman, who added that the marine snail is an excellent model for studying the brain and memory.

Scientists know more about the cell biology of this simple form of learning in this animal than any other form of learning in any other organism, Glanzman said. The cellular and molecular processes seem to be very similar between the marine snail and humans, even though the snail has about 20,000 neurons in its central nervous system and humans are thought to have about 100 billion.

In the future, Glanzman said, it is possible that RNA can be used to awaken and restore memories that have gone dormant in the early stages of Alzheimer's disease. He and his colleagues published research in the journal eLife in 2014 indicating that lost memories can be restored.

There are many kinds of RNA, and in future research, Glanzman wants to identify the types of RNA that can be used to transfer memories.


DNA, RNA, and protein extraction: the past and the present

Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.


Results

Integrity of stored RNA

We analyzed the integrity of the stored RNA samples by Agilent 2100 RIN number. Paired frozen and desiccated aliquots of total human RNA samples were analyzed every two months (except at 10 months) for one year of total storage. The integrity of RNA samples that were frozen at −80°C remained consistent, with average RIN scores ranging from 8.8 to 9.1. The integrity of RNA samples that were desiccated and then stored at room temperature were found to maintain average RIN values of between 8.7 and 9.1 at the five tested time points (Fig. 1A, B). The largest discrepancy in RIN value between two paired samples was 0.8 with the SQ 146 desiccated RNA sample having a 0.8 lower RIN score then its frozen counterpart at month two and month four of storage. The average difference between paired desiccated and frozen sample RIN values was 0.2 with frozen samples having the slightly higher average RIN values (Fig. 1B).

(A) RIN Electrophoretic Analysis of RNA integrity from two representative RNA samples which were desiccated and stored at room temperature (D) or Frozen at −80°C (F) for 12 months. Desiccated samples are shown on the left and frozen samples are shown on the right. The two main peaks in each electropherogram are representative of 18S and 28S ribosomal RNA fragments. (B) The average RIN values with standard error of the mean from all RNA samples measured after the given number of months in storage in a desiccated (D) or frozen (F) state.

RT-qPCR validation of long-term desiccated RNA

After two, six and 12 months of storage, up to 10 patient RNA samples were analyzed from both frozen and desiccated aliquots. At the two and six month time points the majority of the tested desiccated samples expressed TBP at levels close to or exceeding the expression of TBP from the source-matched frozen RNA aliquot. Desiccated-to-frozen TBP expression ratios of one indicate equivalent expression of TBP in the frozen and desiccated aliquots, and ratios above one indicate that there are higher TBP message levels in the desiccated aliquots. The average of the relative TBP expression ratios of the paired desiccated to frozen samples was found to be above one after two and six months of storage. After one year of storage, five paired desiccated and frozen RNA samples were analyzed and only one RNA source had a TBP desiccated-to-frozen expression ratio above one but the average of these five ratios was still within half a standard error of the mean to a value of one (Fig. 2).

Stored RNA samples are analyzed after two, six and 12 months by RT-qPCR for one year. The values depicted are TBP expression levels in each desiccated sample relative to its matched frozen aliquot. Error bars represent standard error of the mean among the paired ratios in each time point.

Over the yearlong study the Ct values for qPCR TBP amplification generally increased across all samples as storage time increased. Average Ct values from desiccated and frozen aliquots of five samples at each time point (four at the two month time point) are shown in Table S1. All RNA samples, whether frozen or desiccated, were found to have a higher Ct value after 12 months of storage compared to after two months of storage. From the two month checkpoint to the year checkpoint the Ct value of the desiccated RNA samples increased on average by 2.85 cycles whereas the corresponding frozen samples showed an increase of 1.85 cycles (Table S1). The trend of increasing Ct number did not hold for each sample, as fluctuating values were seen across the RNA specimens over the yearlong study. All Ct values from frozen and desiccated samples stayed within the range of the upper 20 s to mid-30 s in cycle numbers representing positive TBP expression.

qPCR validation experiments were then conducted using three additional primer/probe sets from the Roche Applied Sciences Universal ProbeLibrary Set. cDNA from RNA which was stored desiccated or frozen for 12 months was analyzed for relative expression of ACTB, GAPD and GUSB message in addition to TBP. Relative desiccated-to-frozen RNA expression level ratio averages were very consistent across the four genes after a year of storage (Fig. S1), and high and low ratio outliers were also consistent across the four genes tested.

RNA sequencing analysis of stored RNA

Total RNA was extracted from two human blood samples. Each sample was analyzed by RNA-Seq after no storage (HD6, MB2026), after 76 days of storage desiccated at room temperature (HD6D, MB2026D) or after being frozen at −80°C (HD6F, MB2026F). Nearly 97% of all sequence tags from fresh, frozen and desiccated RNA aliquots of both samples could be mapped to HG19 (Table 1). Figure 3 illustrates the pair-wise gene expression comparison plots for the different storage methods in these two samples. From these gene expression comparisons, RNA extracted from fresh samples (HD6 and MB2026) and compared to the desiccated (HD6D, MB2026D) or frozen (HD6F and MB2026F) shared high degree of correlations (R 2 = 0.93) (Fig. S2). Furthermore, the gene expressions obtained from desiccated (HD6D, MB2026D) and frozen (HD6F and MB2026F) methods are nearly identical (R 2 = 0.997 and 0.999) (Fig. 3). This indicates that the RNA stored in a desiccated state maintained the same gene expression profiles as the RNA stored in the traditional frozen method.


How RNAi Works

The term RNA interference (RNAi) was coined to describe a cellular mechanism that use the gene's own DNA sequence of gene to turn it off, a process that researchers call silencing. In a wide variety of organisms, including animals, plants, and fungi, RNAi is triggered by double-stranded RNA (dsRNA).

During RNAi, long dsRNA is cut or "diced" into small fragments

21 nucleotides long by an enzyme called "Dicer". These small fragments, referred to as small interfering RNAs (siRNA), bind to proteins from a special family: the Argonaute proteins. After binding to an Argonaute protein, one strand of the dsRNA is removed, leaving the remaining strand available to bind to messenger RNA target sequences according to the rules of base pairing: A binds U, G binds C, and vice versa. Once bound, the Argonaute protein can either cleave the messenger RNA, destroying it, or recruit accessory factors to regulate the target sequence in other ways.

RNAi is widely used by researchers to silence genes in order to learn something about their function. siRNAs can be designed to match any gene, can be manufactured cheaply, and can be readily administered to cells. One can now order commercially synthesized siRNAs to silence virtually any gene in a human or other organism's cell, dramatically accelerating the pace of biomedical research. Furthermore, the ability to turn off expression of a single gene makes RNAi an appealing therapeutic approach to treat infectious diseases or genetic disorders, such as those that result from the inappropriate and undesirable activity of a gene, as in many cancers and neurodegenerative diseases. There are currently several clinical trials testing the safety and effectiveness of siRNA drugs.

RNAi is much more than a research tool. RNAi encompasses an array of ancient and sophisticated cellular mechanisms that regulate a variety of biological functions. Argonaute proteins bind many naturally occurring small RNAs to defend against transposable elements, maintain chromosome structure and stability, and regulate developmental timing and differentiation. For example, microRNAs represent a natural form of developmentally-important siRNAs. Like siRNAs, microRNAs are made by Dicer, but microRNA derive from single-stranded RNAs that fold back on themselves to generate small regions of double-stranded RNA—so called "stem-loops"— instead of the long double-stranded RNA that produces siRNAs. microRNAs can guide Argonaute proteins to repress messenger RNAs that match the miRNA incompletely, allowing one microRNA to regulate hundreds of genes. Humans make more than 500 distinct microRNAs, and the inappropriate production of specific microRNAs has been linked to several diseases. Drugs to inhibit disease-causing microRNAs are now being tested as therapies for several human diseases.


What is RNA?

Let&rsquos begin with the basics. Deoxyribonucleic acid (DNA) is a molecule you may already be familiar with it contains our genetic code, the blueprint of life. This essential molecule is the foundation for the &ldquocentral dogma of biology&rdquo, or the sequence of events necessary for life to function. DNA is a long, double-stranded molecule made up of bases, located in the cell&rsquos nucleus. The order of these bases determines the genetic blueprint, similar to the way the order of letters in the alphabet are used to form words. DNA&rsquos &lsquowords&rsquo are three letters (or bases) long, and these words specifically code for genes, which in the language of the cell, is the blueprint for proteins to be manufactured.

To &lsquoread&rsquo these blueprints, the double-helical DNA is unzipped to expose the individual strands and an enzyme translates them into a mobile, intermediate message, called ribonucleic acid (RNA). This intermediate message is called messenger RNA (mRNA), and it carries the instructions for making proteins. The mRNA is then transported outside of the nucleus, to the molecular machine responsible for manufacturing proteins, the ribosome. Here, the ribosome translates the mRNA using another three-letter word every three base pairs designates a specific building block called an amino acid (of which there are 20) to create a polypeptide chain that will eventually become a protein. The ribosome assembles a protein in three steps &ndash during initiation, the first step, transfer RNA (tRNA) brings the specific amino acid designated by the three-letter code to the ribosome. In the second step, elongation, each amino acid is sequentially connected by peptide bonds, forming a polypeptide chain. The order each amino acid is crucial to the functionality of the future protein errors in adding an amino acid can result in disease. Finally, during termination, the completed polypeptide chain is released from the ribosome and is folded into its final protein state. Proteins are required for the structure, function, and regulation of the body's tissues and organs their functionality is seemingly endless.

Throughout the latter half of the 20th century, we believed that RNA&rsquos primary role was to intermediate between DNA and protein, as we described above. Over the last three decades, those long-held beliefs have been shattered. We have witnessed amazing discoveries with regards to RNA biology, many of which have come from our own labs here at the RTI. In 1998, Andrew Fire and the RTI&rsquos Craig Mello discovered RNA interference (RNAi), in which double-stranded RNA can find and turn off specific genes based on certain sequences (order of the 'words'). For this, they earned the Nobel Prize in 2006! To understand more about RNAi and learn how we are developing this tool into a therapeutic platform, please see: What is RNAi?

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Before beginning your work, your lab should be properly prepared to work with RNA. As mentioned before, RNA can be quite fragile if mishandled and RNases are notoriously difficult to remove. There are four factors to pay attention to when preparing your lab: pH, temperature, metal ions, and RNases.

1. Designate one area for RNA extraction: This becomes especially useful if you’re working in a shared lab. Having just one part of the lab designated to RNA extraction and handling limits sources of RNase contamination.

2. Use disposable RNase free plastic ware: The label on the plastic-ware must explicitly indicate that it’s “RNase-free.” Don’t assume that something is RNase-free because it’s sterile as sterilization doesn’t get rid of RNases. In addition, the pipette tips used should contain a filter to prevent any contamination. (Note: If you would like to use glassware, it should be baked at +180°C to +200°C for at least 4 hours. Autoclaving will not get rid of RNases.)

3. Treat reusable plastic ware with EDTA: If you intend to use reusable plastic ware, it must be soaked (2 hours, +37°C) in 0.1 M NaOH/1 mM EDTA (or absolute ethanol with 1% SDS) to get rid of any metal ions. Then rinse with DEPC-treated water and heat to +100°C for 15 minutes.

4. Gather all your lab equipment: Make sure that all the equipment you will be using are available and easy to access. You don’t want to end up scrambling for a pipette mid-extraction.

5. Clean the lab and equipment: Make sure to clean all benches, pipettes etc. in the RNA extraction area using 100% Ethanol or a commercial RNase inactivating agent. Also, reserve all the chemicals used in RNA extraction and analysis for use in RNA applications only.

6. Always keep the temperature low: All RNA samples should be kept at 0 - 4°C while working with them. That’s why it’s important to ensure that you always have ice on hand before collecting samples or removing them from storage.


RNA Sequencing

RNA sequencing (RNA-Seq) is revolutionizing the study of the transcriptome. A highly sensitive and accurate tool for measuring expression across the transcriptome, it is providing researchers with visibility into previously undetected changes occurring in disease states, in response to therapeutics, under different environmental conditions, and across a broad range of other study designs.

RNA-Seq allows researchers to detect both known and novel features in a single assay, enabling the detection of transcript isoforms, gene fusions, single nucleotide variants, and other features without the limitation of prior knowledge.

Propelling Progress with RNA-Seq

RNA sequencing can have far-reaching effects on research and innovation, transforming our understanding of the world around us.

Propelling Progress with RNA-Seq

Benefits of RNA Sequencing

RNA-Seq with next-generation sequencing (NGS) is increasingly the method of choice for researchers studying the transcriptome. It offers numerous advantages over gene expression arrays.

  • Broader dynamic range enables more sensitive and accurate measurement of gene expression
  • Not limited by prior knowledge - captures both known and novel features
  • Can be applied to any species, even if reference sequencing is not available
  • A better value, often delivering advantages at a comparable or lower price per sample than many arrays
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Learn how RNA-Seq is advancing transcriptome research in various fields, and how gene regulation studies can provide complementary information.

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RNA-Seq is by far the most cited NGS method. This collection contains protocol diagrams, advantages and disadvantages, and related peer-reviewed publications on various RNA-Seq methods featuring Illumina technology.

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How Can I Use NGS to Analyze RNA?

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Key RNA-Seq Methods
MRNA Sequencing

Sensitively and accurately quantify gene expression, identify known and novel isoforms in the coding transcriptome, detect gene fusions, and measure allele-specific expression.

Targeted RNA Sequencing

Analyze gene expression in a focused set of genes of interest. Targeted RNA-Seq can be achieved via either enrichment or amplicon-based approaches.

Ultra-Low-Input and Single-Cell RNA-Seq

Use deep RNA-Seq to examine the signals and behavior of a cell in the context of its surrounding environment. This method is advantageous for biologists studying processes such as differentiation, proliferation, and tumorigenesis.

RNA Exome Capture Sequencing

Achieve cost-effective RNA exome analysis using sequence-specific capture of the coding regions of the transcriptome. Ideal for low-quality samples or limited starting material.

Total RNA Sequencing

Accurately measure gene and transcript abundance and detect both known and novel features in coding and multiple forms of noncoding RNA.

Small RNA Sequencing

Isolate and sequence small RNA species, such as microRNA, to understand the role of noncoding RNA in gene silencing and posttranscriptional regulation of gene expression.

Ribosome Profiling

Deeply sequence ribosome-protected mRNA fragments to gain a complete view of the ribosomes active in a cell at a specific time point, and predict protein abundance.

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Illumina Stranded mRNA Prep

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DRAGEN RNA Pipeline

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Exam Questions on Molecular Biology | Biology

Ans: This hypothesis accounts for the observed pattern of degeneracy in the third base of a codon. According to this hypothesis the third base can undergo with the corresponding first base in the anticodon. The importance of wobble and degeneracy of the genetic code is that cell does not have to synthesize a different tRNA for each of the 61 sense codons. A simple example is that only two different tRNA anticodons are needed to recognize four different glycine codons.

Q.2. What is Shine-Dalgarno sequence? In which groups of microorganisms it is found?

Ans: The Shine-Dalgarno sequence is located before the start sequence of mRNA. This nucleotide sequence allows the mRNA to align with the 30S ribosomal subunit of the bacterial cell. It is about 7 bases upstream earlier) towards the 5 ‘-P end of the AUG start codon on the mRNA and is a polypurine consensus sequence AGGAGG and is referred to as Shine-Dalgarno sequence (Fig. 33.7). It is found in bacterial and archaeal cells.

Q.3. What do the codons UGA, UAA and UAG mean in normal translation?

Ans: In normal translation they mean for “Stop” codons.

Q.4. Why is genetic code said to be degenerate?

Ans: It is because more than one codon can code for the same amino acid.

Q.5. How many termination codons or nonsense codons are there?

Ans: There are only three termination codons also, called “nonsense codons” as they do not code for any amino acid.

Q.6. The codon AGG normally codes for argine but in altered translation it codes for stop. Where does it occur?

Ans: It occurs in human mitochondria.

Q.7. What is odd in the studies made in the mitochondrial DNA (double-stranded circular DNA genome) as one proceeds from lower to higher eukaryotes?

Ans: While one proceeds from lower to higher eukaryotes, the odd thing in the mitochondrial genome is that it gets smaller, e.g., the yeast mitochondrial DNA is five times larger than that of the human mitochondria.

Q.8. What has enabled a given tRNA that sometimes it specifically recognizes several codons?

Ans: The wobble in the base pair at the 5′ end on the anticodon enables the given tRNA to recognize several codons.

Q.9. All newly synthesized bacterial proteins start (initiate) with formylmethionine (may be abbreviated as fmet). How is formyl group removed from the fmet polypeptide in bacteria?

Ans: The formyl group is often removed from formyl-methionine by the enzyme deformylase leaving behind the methionine as the first amino acid in the polypeptide chain.

Q.10. What are genes? Define.

Ans: A gene may be defined as a sequence of nucleotides which specifies a particular polypeptide chain or RNA sequence or that regulates the expression of other genes. The genes which code for proteins are referred to as structural genes or cistrons while the other genes bearing regulatory function are called regulatory genes. The regulatory genes work to control the expression of structural genes. The structural and regulatory genes collectively constitute the genotype which determines the phenotype, i.e., observable structural and functional characteristics.

Ans: A DNA sequence which codes for one or more structural genes (polypeptides) of related function and the DNA sequence which regulates the expression.

Q.12. What controls induction and repression?

Ans: The regulatory genes that produce a regulator protein controls induction (i.e., causing increase in rate of synthesis of an enzyme), and repression (i.e., blockage of gene expression).

Q.13. What is the lac operon?

Ans: The lac operon stands for lactose operon, an operon which contains genes specifying proteins involved in the utilization of (3 -galactosides such as lactose. The lac operon occurs in Escherichia coli at ca. (= about) 8 minutes on the chromosome map. It has the structural: promoter-operator lac Zr-lac Y-lac A. The lac Z gene encodes P-galactosidase, lac Y encodes (3 -galactoside permease and lac A encodes thiogalactoside transacetylase (Fig. 33.8).

Q.14. What is catabolite repression?

Ans: The catabolite repression is the repression of transcription of genes coding for certain inducible enzyme systems by glucose or other readily utilizable carbon sources.

Q.15. What are positive regulators (activators) and negative regulators (repressors)? Describe.

Ans: Bacteria possess many enzymes whose rate of synthesis depends on the availability of external food molecules. These external molecules called inducers and co-repressors usually determine the rate of synthesis of enzymes by controlling the synthesis of their mRNA templates. Inducers and co-repressors act by binding to regulatory proteins referred to as activators and repressors.

Activators are positive regulators because their presence is required for the regulated enzyme to be made while repressors act as negative regulators because their regulatory activity is to prevent the synthesis of proteins. Thus, when lactose is absent the lac operon (lactose operon) repressor prevents synthesis of enzymes that metabolize lactose. However, upon binding an inducer (a molecule related to lactose) the repressor loses this Ability and permits the production of enzymes. The arabinose operon C protein which is an activator causes making of arabinose enzymes on binding to the inducer arabinose.

Q.16. What is palindromic sequence of DNA?

Ans: Literally speaking palindrome is a word that reads the same backward and forward. The palindromic sequence is a region of a nucleic acid that contains a pair of inverted repeat sequences. In a double stranded molecule of DNA such a region shows two-fold rotational (dyad) symmetry or hypemated dyad symmetry if the two IR sequences are separated by another sequence. A double-stranded palindromic sequence can adopt either of two possible formations:

1. A linear structure with inter-strand hydrogen bonding, e.g.,

2. A cruciform structure in which of two strands each forms hairpins by intra-strand hydrogen bonding.

Q.17. Who discovered that X-rays induce mutations?

Ans: Hermann Muller and L.J. Stadler discovered independently in 1927 that X-rays induce mutations.

Ans: A cistron is a gene as defined in terms of the CIS-TRANS TEST, i.e., in a diploid cell or merozygote either of two homologous sequences in a genetic nucleic acid in which two mutations in trans fail to exhibit complete complementation. A cistron may also be defined as the functional unit of genetic inheritance a segment of genetic nucleic acid which codes for a specific polypeptide chain. The term cistron has also been used as a synonym for gene.

Ans: The term recon was coined by S. Benzer. According to him it is a unit of genetic subdivision beyond which recombination does not occur.

Q.20. Define a mutator or mutator gene.

Ans: A mutator gene or mutator is designated as must within which certain mutations cause an increase in the spontaneous mutation rate in other genes, e.g., Escherichia coli mutations in the gene encoding the e subunit of DNA polymerase III (dna Q and mut D alleles) can result in extremely high levels of spontaneous mutations. The mutant alleles may differ from the wild type, e.g., by only one or two amino acid changes in the ε subunit. The mutation may lead to reduced accuracy in the polymerising (nucleotide selection) activity or in the proof reading activity of the enzyme. Some other mutator genes in E.Coli include in the mismatch repair system.

Q.21. What are split genes? Describe.

Ans: The split genes may be defined as the genes that are coded for, by noncontiguous segments of the DNA so that the mRNA and the DNA for the protein product of that gene are not colinear. These are the genes with intervening nucleotide sequences not involved in coding for the gene product.

The split genes have also been regarded as interrupted genes. A structural gene encoding a protein, rRNA or tRNA that contain one too many intervening sequences (introns) that although represented in primary RNA transcript of the gene are absent from the mature RNA molecule (mRNA, tRNA), therefore, do not contribute to the structure of the gene product.

Thus mutation of the RNA transcript of a split gene must involve a process of splicing to delete the intron and join together the remaining sequences called exons. In the case of mRNA the sequence of exons includes the coding sequence of the gene as well as noncoding leader and/ or trailor sequences. Introns themselves are usually noncoding. Most of the nuclear structural genes in higher eucaryotes are split genes.

Q.22. What are overlapping genes?

Ans: The overlapping genes are two or more genes in which part or complete gene is co-extensive with part of another. The genes may be translated in different reading frames or in the same reading frame with different start and/or stop points or different splicing patterns. The phenomenon of overlapping genes maximizes the coding capacity of a genome and can also provide a means for the regulation of expression of genes.

Q.23. The history of DNA world is written in gene sequences. Justify this statement.

Ans: The evolution of the organisms from a common ancestor is represented by a branched pathway known as geological tree (also known as phylogenetic or evolutionary tree). The branching pattern of tree is calculated using the principle of parsimony (based on economy) to determine the minimum number of genetic changes required to derive the sequence of the gene in each organism from a common ancestor. It is sometimes reasonable to assume that highly conserved protein like globin and cytochorme c can be used as a molecular clock to measure how long the species have been diverging from each other.


PrimerDigital

NAsafe is salting-out all proteins media of ammonium sulfate (the saturating concentration) with pH < 5 (see the lyotropic series).

The description of NAsafe (4 M (NH4)2SO4, 10 mM EDTA, 0.1 M Mes, pH 4.6):
combine 40 ml 0.5 M EDTA, 19.5 g Mes free acid, 528 g ammonium sulfate, dissolve in Milli-Q water, bring final volume 1L stir on a hot plate stirrer on low heat until the ammonium sulfate is completely dissolved. Transfer to a screw top bottle and store either at room temperature or refrigerated.

Store NAsafe solution at room temperature. It is guaranteed for 6 months from the date of receipt, if properly stored. If any precipitation of NAsafe solution is seen, heat it to 37°C and agitate to redissolve it.

RNase inactivation is reversible do not rinse NAsafe solution from samples before using. Blot tissues with a wipe, or pellet cells to remove excess NAsafe solution.

Guidelines for use of NAsafe solution
&bull Use NAsafe solution with fresh tissue only do not freeze tissues before immersion in NAsafe solution.
&bull Before immersion in NAsafe solution, cut large tissue samples to &le 0.5 cm in any single dimension.
&bull Place the fresh tissue in 5&ndash10 volumes of NAsafe solution.
&bull Most samples in NAsafe solution can be stored at room temp for 1 week without compromising RNA quality, or at &ndash20°C or &ndash80°C indefinitely.
&bull Do not freeze samples in NAsafe solution immediately store at 4°C overnight (to allow the solution to thoroughly penetrate the tissue), remove supernatant, then move to &ndash20°C or &ndash80°C for long-term storage.

Most samples can be stored at 25°C in NAsafe solution for up to 1 week without significant loss of RNA quality. After 2 weeks at 25°C, RNA generally appears slightly degraded (marginally acceptable for Northern analysis, but still of sufficient quality for nuclease protection assays or RT-PCR analysis).

The lyotropic series salts
Some anions and cations have been noted to be effective in the order of salting-out to salting-in effect:

NH4 + > Na + > K + > Li + > Rb + > Cs + > H + > Mg 2+ > Ca 2+ > Urea > [CH6N3] + (guanidine)

OH - > SO4 -2 > HPO4 -2 > F - > citrate -3 > tartrate -2 > CH3COO - > Cl - > Br - > NO -3 > SCN - > ClO4 - > I -

These series known as lyotropic or Hofmeister series were related to a special physicochemical property of the ion called &ldquolyotropy&rdquo. Ions on the left such as strongly hydrated but weakly lyotropic anions of SO4 -2 are called kosmotropes, while the weakly hydrated ions, such as lyotropic anions of ClO4 - are referred to chaotropes.

(NH4)2SO4 and Na2SO4 is showing the greatest effect on the salting-out (precipitation) of proteins guanidinium thiocyanate is showing the greatest effect on the salting-in (chaotropic agent and denaturant) of proteins.

Guanidinium salts and urea are strong chaotropic salts that disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions resulting in a drastic effect on other solute molecules. For example, urea, when dissolved in water, disrupts the secondary, tertiary, and quatemary structures of proteins, and subsequently causes dissociation of proteins from RNA. Guanidinium salts and urea dissolve in water through endothermic reactions. Both guanidinium salts and urea are considered to be strongly chaotropic salts as defined by the Hofmeister series, a widely used system that ranks cations and anions according to relative chaotropic strength.

It has been believed both experimentally and theoretically that the effectiveness is more pronounced for anions than cations, which is classically used to describe the capacity of an ion to enhance or weaken the hydrogen-bond structure of water molecules, or, in other words, to enhance or reduce the solubility of a solute, respectively.

The overall salt effect depends on the nature of salts, surface, substrate, temperature and concentration. Although salt effects are very complex and quite different in different systems, lyotropic salts can be employed to shed light on the presence of molecular interactions in polymer systems.

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