Why is knowing GC content important?
The GC Content as a Main Factor Shaping the Amino Acid Usage During Bacterial Evolution Process. Understanding how proteins evolve is important, and the order of amino acids being recruited into the genetic codons was found to be an important factor shaping the amino acid composition of proteins.
Why is high GC content good?
GC-rich DNA sequences are inherently more stable than sequences with a low GC content. For PCR, this means that the higher the GC content, the higher the melting point of the DNA. This is why Thermus thermophilus, an extremophile that needs to tolerate very high environmental temperatures, has a GC-rich genome.
How does GC content affect sequencing?
GC-content normalization is designed to reduce the dependence of gene-level read counts on sequence composition within a lane. However, other technical effects, such as between-lane differences in sequencing depth, can strongly bias differential expression results.
Why is GC content important in PCR?
DNA templates with high GC content (>65%) can affect the efficiency of PCR due to the tendency of these templates to fold into complex secondary structures. This is due to increased hydrogen bonding between guanine and cytosine bases, which can cause the DNA to be resistant to melting.
What happens if GC content is high?
High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques.
What does low GC mean?
While many Firmicutes stain Gram-positive, some do not. In fact, some Firmicutes have no cell wall at all! They are called "low G+C" because their DNA typically has fewer G and C DNA bases than A and T bases as compared to other bacteria.
Why is high GC content bad for sequencing?
High GC regions will have lower coverage because they relate to 'theremodynamically unfastened' regions that require more energy (heat) in order to separate the strands. If the strands cannot be separateed, they acn neither be amplified in cclonal amplification and cannot be sequenced.
How does GC content affect gene expression?
The GC content of a gene region can impact its coverage, with regions having 50–60% GC content receiving the highest coverage while regions with high (70–80%) or low (30–40%) GC content having significantly decreased coverage [14].
What is considered low GC content?
The GC-content of most species does tend to hover near 50%. However, coding regions of the genome have a tendency to contain a higher percentage of guanine and cytosine; these areas are called GC-rich, in contrast to areas of GC-content below 50%, which are called GC-poor.
Why is DNA with a higher GC content more stable?
From the base-pairing diagram, we can see that the G-C pair has 3 hydrogen bonds, while the A-T pair has only 2. Therefore, the G-C pairing is more stable than the A-T pairing. Thus, strands with more G-C content have more hydrogen bonding, are more stable, and have a greater resistance to denaturation.
How GC content helps for classification of bacteria?
In turn, these interactions seem to be important in conferring stability to higher order structures of DNA and RNA transcripts (11, 20). In bacteria, for example, an increase in GC content correlates with a higher temperature optimum and a broader tolerance range for a species (21, 22).
Which bacteria have high GC content?
Nitrogen-fixing aerobic bacteria have higher genomic GC content than non-fixing species within the same genus.
Why is DNA with a higher GC content more stable?
From the base-pairing diagram, we can see that the G-C pair has 3 hydrogen bonds, while the A-T pair has only 2. Therefore, the G-C pairing is more stable than the A-T pairing. Thus, strands with more G-C content have more hydrogen bonding, are more stable, and have a greater resistance to denaturation.
Why is high GC content bad for sequencing?
High GC regions will have lower coverage because they relate to 'theremodynamically unfastened' regions that require more energy (heat) in order to separate the strands. If the strands cannot be separateed, they acn neither be amplified in cclonal amplification and cannot be sequenced.
How does GC content affect gene expression?
The GC content of a gene region can impact its coverage, with regions having 50–60% GC content receiving the highest coverage while regions with high (70–80%) or low (30–40%) GC content having significantly decreased coverage [14].
Why is it important to have a relatively high proportion of G and C bases in your primer?
The presence of G and C bases at the 3′ end of the primer—the GC clamp—helps promote correct binding at the 3′ end because of the stronger hydrogen bonding of G and C bases. GC bonds contribute more to the stability—i.e., increased melting temperatures—of primer and template, binding more than AT bonds.
Why are GC rich regions important?
Moreover, the GC-rich regions have pivotal importance in techniques like PCR or DNA sequencing. Unlike the AT-rich regions, the GC content may decrease the specificity and efficiency of the PCR or DNA sequencing.
Why are GC templates higher in PCR?
In the PCR, higher GC templates increase the chances of non-specific bindings and consequently the chances of false-positive results. Care must be taken while selecting the PCR template DNA and designing primers.
What is a CPG island?
Broadly, the higher GC-rich repetitive areas are denoted as ‘CpG island’ that also have a definite role in the development of disease. Huntington’s disease is the classic example of a change in the number of CAG repeats of the HTT gene.
What are the GC-rich domains in the human genome?
The huge regions in the human genome have GC-rich domains often known as “isochores” and took part in constructing some vital genes too. Cytologically, the region consists of the GC base pairs stains darker than the AT-rich regions.
How much GC do humans have?
We have enlisted the GC-content of different organisms which shows that humans have 39.7% of this part. Importantly, it’s present not only in heterochromatin but also in euchromatin and so is an important part of genes too.
Is the genome GC rich?
Our genome is huge and has many mysteries, the GC-rich regions are one among those, Here in the present article, I will explain the importance of the GC bases in our genome as well as in techniques like PCR or sequencing.
What is the GC content of an organism?
The GC-content of organisms is a highly variable trait. For example, in bacteria GC-content can range from lower than 25% to higher than 75% (Lynch, 2007; Bentley and Parkhill, 2004; Sueoka, 1962 ). Variation in nucleotide content is observed in a correlated manner across all types of sites, including nonprotein-coding regions, synonymous sites and nonsynonymous sites ( Hershberg and Petrov, 2009, 2010, 2012; Figure 1 ). For many years it was thought that the nucleotide composition of a genome is determined by mutational biases (e.g., Andersson and Sharp, 1996; Chen et al., 2004; Hershberg and Petrov, 2008; Shields, 1990; Muto and Osawa, 1987; Sueoka, 1962 ). In other words, it was thought that GC-rich genomes arose form GC-biased mutation patterns, while AT-rich genomes arose from AT-biased mutation patterns. However, recently it has been shown that mutational biases in themselves cannot explain nucleotide content variation, as for both AT-rich and GC-rich genomes, mutations are universally more likely to occur from G/C to A/T ( Hildebrand et al., 2010; Hershberg and Petrov, 2010 ). The causes of variation in GC-content have therefore not been satisfactorily resolved. In the end, the GC-content of an organism is likely determined by a complex combination of neutral and selective processes that can cause the substitution patterns of the genome to be biased either toward GC or toward AT ( Duret and Galtier, 2009; Foerstner et al., 2005; Hershberg and Petrov, 2010; Hildebrand et al., 2010; Reichenberger et al., 2015 ).
What is the percentage of GC?
The GC content (percentage) is the number of GC nucleotides divided by the total nucleotides.
How to find the G+C content of a sample?
The G+C content is estimated from the ratios of dThd and dGuo. To determine the G+C content of a sample, first the constant Y must be determined by chromatography of standard DNA. The constant Y is defined by Eq. (1):
How to use additives in PCR?
The best approach in using an additive, to troubleshoot a problem or improve PCR quality, is to make a titration of several concentrations within the recommended concentration range (as listed in the Table earlier ). For instance, if high GC% is a problem, then try adding DMSO to the PCR reaction, make 2, 4, 6, and 8% (final DMSO concentration) titration points and check which one gives best results for the target of interest.
How many genes are encoded in the C. reinhardtii genome?
Merchant et al. (2007) tabulated 259 genes encoding tRNAs in the C. reinhardtii genome sequence. Detailed information can be obtained in the supplementary material for this paper.
Why is the chemical environment less aggressive?
Alternative explanation, however, is that in long-lived species, the chemical environment is likely less aggressive (e.g., because this ensures slower damage accumulation presumably required for slower aging). As we have seen earlier ( Section 2 ), the most pressing damage-related mutation in mtDNA is the G to A change likely resulting from deamination of C. Less aggressive environment may mean lower mutational pressure on the G, which could account for the observed higher GC content in long-lived species. Note that in this explanation, mtDNA content is considered merely a marker of chemical damage not a causative factor.
Why do gene modelers predict more accurately in low GC regions?
Most gene modelers predict more accurately in low GC regions because they strongly rely on hexamer frequencies to discriminate between coding and noncoding regions ( Burset and Guigo 1996 ). In fungal genomes the G+C content varies greatly from 33% for Candida albicans to 57% in P. chrysosporium.
What is GC in chemistry?
Gas chromatography (GC) is an analytical technique used to separate and analyze samples that can be vaporized without thermal decomposition. Sometimes gas chromatography is known as gas-liquid partition chromatography (GLPC) or vapor-phase chromatography (VPC). Technically, GPLC is the most correct term, since the separation of components in this type of chromatography relies on differences in behavior between a flowing mobile gas phase and a stationary liquid phase .
How is GC used in chemistry?
GC is used as one test to help identify components of a liquid mixture and determine their relative concentration. It may also be used to separate and purify components of a mixture. Additionally, gas chromatography can be used to determine vapor pressure, heat of solution, and activity coefficients. Industries often use it to monitor processes to test for contamination or ensure a process is going as planned. Chromatography can test blood alcohol, drug purity, food purity, and essential oil quality. GC may be used on either organic or inorganic analytes, but the sample must be volatile. Ideally, the components of a sample should have different boiling points.
What is the peak of a chromatogram?
Usually, the first peak is from the inert carrier gas and the next peak is the solvent used to make the sample.
What is make up gas?
When the support gas is called "make up gas", it means gas is used to minimize band broadening. For FID, for example, nitrogen gas (N 2) is often used. The user manual that accompanies a gas chromatograph outlines the gases that can be used in it and other details.
Why is the tube long?
The tube is long to allow for a better separation of components. At the end of the tube is the detector, which records the amount of sample hitting it. In some cases, the sample may be recovered at the end of the column, too. The signals from the detector are used to produce a graph, the chromatogram, which shows the amount ...
What factors affect the rate at which a compound progresses down the column?
Other factors that affect the rate at which a compound progresses down the column (called the elution time) include polarity and the temperature of the column. Because temperature is so important, it is usually controlled within tenths of a degree and is selected based on the boiling point of the mixture.
What is the gas used in FID?
For FID, for example, nitrogen gas (N 2) is often used.
Which processes drive GC content evolution in the human genome?
Both selective and neutral processes drive GC content evolution in the human genome.
Does GC content evolve over time?
Interesting question. The GC-content seems to evolve over time and it also seems that the GC-content of coding regions is higher than for the surrounding non-coding regions (see reference 1). If there is a specific function for this higher GC-content or not is (if I understand this right) debated among the groups which do research in this field. Have a look at the references (and probably also their references) to decide on this:
What is GC Content?
GC content is usually calculated as a percentage value and sometimes called G+C ratio or GC-ratio. GC-content percentage is calculated as Count (G + C)/Count (A + T + G + C) * 100%.
How does GC affect DNA?
The GC content affects the stability of DNA. The GC content affects the secondary structure of mRNA. The GC content affects the annealing temperature for template DNA in PCR experiments.
What is GC used for?
GC is a widely used technique across most industries. It is used for routine analysis through to research, analysing a few to many hundreds (or thousands with GC x GC) of compounds in many different matrices, from solids to gases. It is a robust technique and is easily hyphenated to other techniques including mass spectrometry.
What is the most common problem in GC?
The most common problem in GC is leaks. The mobile phase is a gas and flows throughout the system, therefore the correct installation of parts and consumables is important along with regular leak checking.
What is gas chromatography?
Gas chromatography (GC) is an analytical technique used to separate the chemical components of a sample mixture and then detect them to determine their presence or absence and/or how much is present. These chemical components are usually organic molecules or gases. For GC to be successful in their analysis, these components need to be volatile, usually with a molecular weight below 1250 Da, and thermally stable so they don’t degrade in the GC system. GC is a widely used technique across most industries: for quality control in the manufacture of many products from cars to chemicals to pharmaceuticals; for research purposes from the analysis of meteorites to natural products; and for safety from environmental to food to forensics. Gas chromatographs are frequently hyphenated to mass spectrometers (GC-MS) to enable the identification of the chemical components.
How do you read a chromatogram and what does it tell you?
Figure 2: Chromatogram output from a GC or GC-MS. Credit: Anthias Consulting.
What is the final step in GC?
The final step is the detection of the analyte molecules when they elute from the column. There are many types of GC detectors, for example: those that respond to C-H bonds like the flame ionization detector (FID); those that respond to specific elements for example sulfur, nitrogen or phosphorus; and those that respond to specific properties of the molecule, like the ability to capture an electron, as is used with the electron capture detector (ECD).
What is the x axis of a GC?
The x-axis is the retention time, taken from the time the sample was injected into the GC (t 0) to the end of the GC run. Each analyte peak has a retention time measured from the apex of the peak, for example t R. The y-axis is the measured response of the analyte peak in the detector. The baseline shows the signal from the detector when no analyte is eluting from the column, or it is below the detection limit. The baseline response is a mix of electrical noise (usually low) and chemical noise, such as impurities in the carrier gas, column stationary phase bleed and system contamination. Hence, if the baseline is higher than it should be, it is an indication of a problem or that maintenance is required. Various measurements can be taken from the peak, such as width at the baseline, width at half height, total height and area. The latter two are proportional to the concentration, however it is the area that is used for quantitation as it is less affected by band broadening. The measurements can be used to calculate the extent of band broadening, the spread of the analyte molecules on the column. Narrower, sharper peaks give better sensitivity (signal to noise ratio) and better resolution (peak separation). The peaks shown are Gaussian, however peak tailing (the right side of the peak is wider) indicates activity or a dead volume in the system, whereas a peak fronting (the left side of the peak is wider) indicates the column is overloaded. Accurate measurements are affected by the number of data points across a peak, with an ideal number being 15-25. Too few, makes the peak look like a child’s join-the-dots drawing, affecting peak area, resolution and, with GC-MS, deconvolution. Too many reduces the signal to noise, reducing sensitivity. For GC-MS data, each data point is a mass spectrum, the third dimension of data.
What is the process of vaporizing a mixture in a GC?
After injection into the GC inlet, the chemical components of the sample mixture are first vaporized, if they aren’t already in the gas phase. For low concentration samples the whole vapour cloud is transferred into the analytical column by the carrier gas in what is known as splitless mode. For high concentration samples only a portion of the sample is transferred to the analytical column in split mode, the remainder is flushed from the system through the split line to prevent overloading of the analytical column.
