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Wednesday, 22 October 2014
One anxiety replaces another... Part 1
I am sitting here with my anxiety/panic attack kit.
Not much in it to be honest. I have my phone, my laptop, a bottle of water and some wet wipes. That's about it really. I have just taken a couple of my beta blockers and I am all set to ride the waves of anxiety.
I am alone. And right now, that's the way I want it to be. I can think relatively clearly and if someone was here having a conversation with me I would probably be quite irritable and snappy.
I wrote last time about my separation anxiety. I had feared that by writing about it I would suddenly make the problem much more real, but at the end of the day, I knew the feelings I was experiencing needed to be addressed.
After the blog the anxieties continued whenever Nathan had any appointments or when he had to go somewhere out with my comfort zone (without me).
The stress this was causing me was unbelievable. In my negative, anxiety riddled mind, I had made the situation much much bigger and scarier than it needed to be. The outcome of the situation because of my messed up perception, was bleak to say the least.
Nathan was due to regularly attend his nursery in another town that I cant get to. He was also due to attend several hospital appointments in the same town. Me going to these places wasn't an option, and so I would need to face the separation anxiety on a regular basis.
My stress and anxieties all came to a head during this time and I felt ill. Really really crappy! My head was firing all the unhelpful negative thoughts around all day. The physical symptoms were constant. Churning stomach, nausea, loss of appetite, the feelings of utter dread. I was finding it hard to focus on day to day tasks. My head was like a sieve. I was permanently nervous and jumpy. Short tempered and never off the toilet!
I missed appointments, made excuses. I avoided. He went to some and I would feel I done well that day, and then he would miss others and I would feel like the worlds worst and most selfish mum ever.
My poor mother, who takes Nathan to all his appointments in my place, she was worried sick. She worries about me as her daughter, but also worries that I wont get a grip on this and her grandson will be left to suffer.
Don't get me wrong. Nathans physical health is all perfect so its nothing too serious. But he needs his behavioural issues looked at so it doesn't affect him later in life. For every missed appointment I was wracked with guilt. Was I jeopardising my own sons future? As a mother, it is your natural instinct to put your child first, no matter how dreadful you feel. So how come I was putting me first? How come I was avoiding these appointments and choosing the easy option that day? A day where I didn't need to feel the anxiety and the discomfort?
Had I faced the appointments I would have felt awful. I would need to go through the anxieties I'm filled with when he is gone, but I would get through it and feel a sense of achievement that I had done the right thing. And that night, I would be relaxed and calm. Instead I choose to avoid the situation, to dodge the unpleasant feelings I would go through. And for an hour I might feel ok, but soon the guilt would kick in. And then the thoughts..... 'If you don't face this now you never will. If you don't address this problem it is never going to go away. You are affecting your sons chance in life. You are creating problems for your son. What If he ends up being like you. What if he ends up with these anxieties. The nursery and the hospital are going to start looking at you. You are not good for Nathan and so your probably going to lose him. They will take him off you' And then I would be a nervous wreck anyway. No relaxing and calm night.... just a permanent state of discomfort and stress.
Broken sleep, weight loss, (or weight gain if your a comfort eater), more anxiety. To be honest, as I read what I have written so far it doesn't paint an accurate description of the utter despair and hopelessness you feel at times like this. Your whole day is dark, your how thought process is about that ONE issue. Whatever the issue is, it is the cause of a multitude of mental and physical reactions. You feel completely and utterly lost.
So I reached out and I got help.
I called my doctor and told the truth. And although I was terrified that I sounded like an irrational nutcase I just had to be honest. I needed someone professional to tell me I wasn't going to lose my son. That's the size the issue had become in my head and I was a bit of a wreck for a week.
It was my doctor who changed the dosage of the beta blockers. What I was taking was apparently just not enough. Apparently I just needed 'a little help'. My beta blockers basically stop some of the physical symptoms. They keep your heart at a nice normal rate and you lose the feeling that its about to burst out of your chest. The churning stomach disappears, the shakes, the dry mouth etc etc. The thoughts are still there but your body doesn't physically react to them in the same way. (Let me stress it doesn't take them away COMPLETELY, otherwise we would all be cured, but it does take the edge off). I was also referred to a mental health team for some support.
I should explain that I am at my happiest and most relaxed when I am at home with my son. I don't really have time to be anxious as he is so demanding.
One day he was going out with his gran and he had only just left.... I was never THAT great when he went away with other people as my little buddy and security blanket was leaving me alone for a day. It felt weird. But he needs to live a normal life. So off he went. But on this particular day, when he left, I had a huge panic attack. And unlike the ones I had controlled in the past, I felt I couldn't get it to stop. This was my mistake! Had I waited just a little longer, the panic would have passed. But I jumped and I ran. I got my son from the bus stop before they had even left. I was shaking like a leaf, I felt like the worst mother ever, but I didn't care. I just needed those sensations to STOP! And when Nathan was in my arms, they did. BAD DECISION. That day I sowed a very nasty little seed.
My fear is that when Nathan leaves... in that first 10 minutes, I will suffer the mother of all panic attacks. And the only way it will stop is if Nathan is with me. If he is out of my comfort zone, then I cant get to him to make the feelings stop. If he is with someone else he wont be back QUICK enough to make the feelings stop... so ill be suffering the most horrendous feelings you can experience with nothing to make it go away.
Is that reality? Is that REALLY what would happen? No of course its not. But it doesn't stop my head from thinking that way. I hope I'm not losing the agoraphobic readers with the fact I've moved onto separation anxiety, but the reality is its all linked. Its all about anxiety. Its all about control and its all about how we think. And so although my agoraphobia is still very present.... this is just my biggest issue at the moment.
So... how do beat this problem? Yeh... you face it. Dead Simple. (hahahaha)
I tried facing it!!! It didn't make it easier the next time. It didn't fix it! Results weren't quick enough and so I panicked even more. Realistically this problem was gonna take time to beat and it couldn't disappear by facing one event. But I would get through 1, 2 then cancel the 3rd. Id manage the 4th then cancel the 5th and 6th. It was never gonna work. But there were days that I literally felt that I had no fight in me and I just couldn't do it!
I had about a week and a half with no nursery and no appointments. Bliss! I decided to relax and use that time to be calm. Get my head together again. Sleep lots, eat lots. Get myself back into a good place and be ready for it all starting again.
Monday this week arrived and BOOM there was the familiar anxious thoughts and feelings. And all because I knew it was time to get back to reality and face life. With 2 appointments and 2 days at nursery all on my first week of facing the problem, I knew I was in for a rough time.
So I am sitting here with my anxiety/panic attack kit.
Not much in it to be honest. I have my phone, my laptop, a bottle of water and some wet wipes. That's about it really. I have just taken a couple of my beta blockers and I am all set to ride the waves of anxiety.
Posted by Lynn at 14:06 11 comments
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WTF Of The Week: Asian Teacher Abuses Children During Class Pictures!
Some asian teacher goes around pushing. and slapping kids around while taking class photos
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Group 1 Project - Fluorescent-PCR
- 1 Introduction
- 2 Basic Concept of DNA
- 3 Development of PCR
- 4 Principles of Fluorescent-PCR Procedures
- 5 Comparison against Conventional PCR
- 6 Clinical Applications of Fluorescent-PCR for Diagnosis
- 7 Fluorescent-PCR Applications in Cell Biology Research
- 8 Glossary
- 9 References
Genetic expression has established the basis of clinical diagnosis and molecular analysis. Any alterations of genetic expression may result in the development of a disease state or abnormal cellular process. Fluorescent polymerase chain reaction (abbreviated as fluorescent-PCR) has been an efficient analytical method that could detect genetic material in an organism with high precision. When genetic material is present in limited amounts, DNA or RNA could be amplified exponentially to a substantial level for detection in a simple enzymatic reaction. Gene amplification accounts for the high sensitivity of PCR where single copies of genes are analyzed. PCR is also characterized by its high selectivity and speed. Therefore, fluorescent-PCR is a diagnostic test that provides simplicity, accuracy, reliability and performance. These characteristics justify the extensive use of fluorescent PCR for genetic screening and analysis in medical research.
Basic Concept of DNA
Amplification of genetic material by PCR is developed on the basis of understanding the DNA biology. Comprehending the basic concept of DNA structure will enhance our appreciation of the PCR technique for current molecular biological research.
DNA has a double-helical structure that constitutes two anti-parallel strands intertwined with one another. Each DNA template strand is a polymer of subunits called the nucleotides. A nucleotide subunit is composed by a phosphate molecule, deoxyribose sugar and a nitrogenous base. The deoxyribose sugar molecules are linked together by the phosphate groups that formed the backbone of the DNA strand. The linkage is known as 3'-5' phosphodiester linkage that connects 3'C of sugar molecule to the 5'C of the adjacent nucleotide subunit.
Deoxyribose sugar molecule is covalently bonded to one of the nitrogenous bases (Adenine, Guanine, Cytosine and Thymine). Adenine (A) and Guanine (G) are double-ringed bases known as purines; while Cytosine (C) and Thymine (T) are the single-ringed bases known as pyrimidines.
The double-helical DNA structure is constructed by two DNA strands oriented in opposite directions and held together by hydrogen bonds. Adenine and Thymine can form a base pair with two hydrogen bonds; and Guanine and Cytosine form a base pair by three hydrogen bonds. These base pairings occur exclusively either between A-T or G-C; therefore, they are called complementary base pairs. The complementary base-pairings contribute to selectivity and specificity of the PCR technique, where a targeted sequence is amplified by initiating DNA extension with primers of complementary gene sequences. In the double-stranded DNA, interaction energies between the complementary bases arise from the hydrogen bondings and the hydrophobic interactions between the neighbouring stacks of base pairs[1] . In the denaturation phase of thermal cycling, these hydrogen bonds are broken to generate single-stranded DNA by high temperatures. However, renaturation can occur as the two single polynucleotide chains unite to form a double-helix where the original hydrogen bonds can reform [2]. This accounts for the annealing of the primers to the complementary sequences of the template strand when the temperature is lowered.
Development of PCR
Since the early 1990s, PCR has predominantly become the basic tool for application in molecular biology. As it progressed to mid 1990s, PCR was used as a diagnostic and screening tool for genetic diseases.
Before PCR was introduced, molecular cloning allowed molecular biological research and studying of genetic structures. The efficiency of this method relied on the DNA replication of plasmids and vectors in cellular division. Researchers recognised that molecular cloning was laborious and possessed low selectivity. Thus, it was difficult to isolate specific DNA from cells in biological specimens.
Development of PCR has revolutionized the procedures of studying molecular biology. This DNA amplification procedure was initiated by Kary B. Mullis and his team from Cetus Corp in 1984[3]. As compared to molecular cloning, PCR amplified DNA via a in-vitro setting instead of in-vivo. Since then, PCR has accelerated the analysis of genetic data. Since its discovery in 1983, Basic PCR has generated large quantities of DNA sequences if the DNA sequences of the primer molecules were known. Primer's DNA sequences would bind to complementary DNA sequences on template strand for amplification. The limitation of this method is the need to know the DNA sequence at both ends of targeted template strand to synthesize the primers.
In the early 1990s, anchored PCR was developed by Gail Martin and Mark Davis of Stanford. This method overcame the limitation in the basic PCR. Anchored PCR employed the use of one primer and another "anchor" primer binding to a sequence artificially linked by unknown an sequence of target. Another strategy developed by Washington University was inverse PCR. DNA template strands were cleaved by restriction enzyme and annealed at each ends to form circular DNA. Synthesis of primers were based on the ends of the known sequence. At each template strand, DNA is transcribed from one primer site to another. This method generates linear DNA molecules where DNA sequences are anti-parallel to the template strand in the first round of amplification. Subsequently, ordinary PCR will proceed.
DNA samples in early PCR experiment were amplified by the Escherichia coli DNA polymerase 1 at the temperature of 37°C. The outcome was incomplete pure target products determined by gel electrophoresis. Isolated heat-resistant DNA polymerase from Thermus aquaticus permits the annealing and extension of DNA at higher temperatures. This DNA polymerase is not denatured at 95°C and it works optimally at 72°C. Non-complementary annealing of the primer and template strand is reduced to negligible level.
Basic PCR Timeline
1971 – Gobind Khorana described a basic principle of replicating a piece of DNA using two primers. Progress was then limited by primer synthesis and polymerase purification issues.
1976 – Taq polymerase was discovered (from Thermus aquaticus) which paved the way for PCR concept. Taq is stable at high temperatures and remains active after DNA denaturation, eliminating the need to replace the DNA polymerase after each cycle of DNA replication.
1983 – Kary Mullis at Cetus Corporation conceived a way to start and stop a polymerase's action at specific points along a single DNA strand. Mullis also realised that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified.
1985 – Science publishes the first paper on PCR.
1989 – PCR "explosion" can be seen as a result of a combination of the improvements and optimising of the methodology and the introduction of new deviations on the basic PCR concept.
1993 – Kary B Mullis receives a noble prize in chemistry for inventing the concept of PCR.
Present – Many variations have built upon the fundamental PCR method, including that of Fluorescent PCR[4].
Principles of Fluorescent-PCR Procedures
Multiple copies of a desired DNA sequence could be amplified through the polymerase chain reaction technique. The sensitivity of this technique is enhanced by performing hybridization of a fluorescent probe to the PCR products. With a fluorescent detector, the PCR products are analyzed based on the fluorescence intensity.
Polymerase Chain Reaction
Although fluorescent-PCR may be the topic of interest, it is relevant to understand the underlying mechanism of the conventional PCR, as fluorescent-PCR is an extension of this fundamental process. The basic principles of amplification in PCR evolved from the knowledge about DNA replication, denaturation and renaturation.
Amplification of the genetic material requires DNA polymerase, two oligonucleotides primer (where each primer is complementary to one strand of parenteral DNA template) and repetitive cycles at three different temperatures known as thermal cycling[5].
The three processes in thermal cycling are: denaturation, annealing and extension.
- Denaturation is the process of heating the DNA duplex to a temperature of 90-95°C. At a high temperature of 90°C, the hydrogen bonds between the complementary strands of DNA helix are broken. Subsequently, two single-stranded of DNA are generated.
- Annealing occurs as the temperature of the process is reduced to 50-65°C. As the reaction is cooled down to 50°C, hydrogen bonds form between the bases of oligonucleotide primers with the DNA template strand. The primers anneal to the complementary DNA sequence on the single-stranded DNA template that begins at the 5' end.
- Extension of the new DNA strand require the temperature to be raised to 72°C. The new DNA strand is extended by Taq DNA polymerase, an enzyme that polymerizes the additional deoxy-nucleotides (dNTP) in a DNA sequences that are complementary to the DNA template. Taq DNA polymerase functions optimally at 72°C, with magnesium in the PCR buffer facilitating the reaction.
One cycle comprises of denaturation, annealing and extension. For substantial genetic material to be analyzed, 25-35 cycles are performed in PCR. DNA strands are amplified exponentially, where the number of DNA strands can be quantified by this formula, 2n, where n denotes the number of thermal cycles.
Fluorescent Analysis
When the amplification of genetic material is completed, the quantity and molecular size (in bps) of amplified products could be determined by gel electrophoresis and fluorescent analysis. When fluorescent probes are used, it would sensitize the analytical technique and allowing visualization through the emission of fluorescence signal.The processes involved in fluorescent analysis are fluorescent labelling, gel electrophoresis and detection of fluorescence for quantification.
1) Fluorescent-labelling
Fluorescence probes are added to the amplified genetic molecules after PCR. Examples of fluorescent probes include MB-Green and MB-Red [6]. Fluorescent-labelled primer is an alternative for detection in quantification. Fluorescent probes are used to identify the presence of specific genetic sequence in PCR products. If the fluorescent probe has a complementary DNA sequence to the wild-type PCR product, any mutations present in the DNA sequencing will disrupt the hybridization between the probe and amplified product. Different fluorophores in the fluorescent probes serve as markers for multiple target genes by variation in colour coding. Presence of mutations and multiple PCR products could be detected simultaneously after PCR is completed.
- In the labelling process, fluorescent probes or primers are added as one of the components in the PCR solution.
- Before the amplification process, the probes were added to the solution. In addition, oligonucleotides (dGTP, dTTP, dATP, dATP), Taq ploymerase, magnesium chloride and Tris solution are other essential constituents in the PCR solution.
- PCR is performed in a thermal cycling for amplification at three different temperatures then incubated at room temperature.
- Fluorescent probes has provide the benefits of in-situ hybridization where the amplification and labelling are performed in the same well.
- The principle of the fluorescent labelling is dependant on the structure of the fluorescent probes and intrinsic nuclease activity of Taq DNA polymerase. These probes will initially be hybridized to the target DNA sequence before cleavage by Taq DNA polymerase during amplification.
- Fluorescent probes are oligonucleotides that has a fluorescent dye at the 5' end and a quencher (E.g. Dabcyl or rhodamine) at the 3' end. These oligonucleotides exist in stem-loop structures where hydrogen bonds are formed between the complementary bases within the same oligonucleotide strand. Upon excitation by irradiation at certain wavelength, the fluorescent dye would emit fluorescence through the resonance energy transfer[7]. If the quencher is in close proximity to the fluorescent dye, the fluorescence emission will be quenched. During extension phase, the hybridized probe will be cleaved by Taq DNA polymerase by its intrinsic nuclease activity from 5' to 3' end. Both fluorescent dye and quencher group will be released from the probe after cleavage. Fluorescent dye is no longer quenched. Subsequently, an increase in fluorescence intensity will be observed, where the degree of quenching is inversely proportional to the distance between the fluorescent dye and quencher. Therefore, amplifications of PCR products are quantified by the elevation of fluorescence signal.
2) Gel Electrophoresis
Gel electrophoresis is used for separation of the amplified genetic product according to its molecular size for quantification in a genescanner. Molecular size is expressed as the number of nucleotide base pairs. In addition, gel electrophoresis can be used to distinguish the intended amplicon from the false amplicon based on the molecular size[8]. Unintended amplicons may occur by the hybridization between the primers that generate a false signal. The separation of PCR products is performed on an agarose or polyacrylamide gel immersed in an electrophoretic buffer that maintains the pH at a consistent value. The degree of DNA fragment separation can be adjusted by varying the concentration of the agarose or polyacrylamide gels. PCR products separate once they are subjected to the electrical field generated from the polarities by the electrodes.
- For conducting gel electrophoresis, the PCR products or DNA sample are initially diluted in Tris solution and EDTA. The diluted PCR products is mixed with the loading buffer that may be constituted by a density gradient agent (E.g. glycerol, sucrose or Ficoll), EDTA and tracking dye (E.g. xylene cyanol, bromophenol blue or orange G). In gel electrophoresis for conventional PCR, ethidium bromide as a fluorescent dye is used for staining nucleic acids for visualisation with a UV transilluminator. In fluorescent- PCR, a fluorescent signal is detected upon excitation which obliterates the use of ethidium bromide. Amplified DNA samples are loaded into the sample wells of agarose gel by a pipette. Beside the PCR products, a DNA ladder is also electrophoresed on the agarose gel at a designated voltage.
- The main principle of gel electrophoresis is utilising both electric field and the porosity of the electrophoresis gel to separate DNA molecules. Nucleic acids possess negative charges from their phosphate backbone. The electrodes (cathode and anode) generate an electric field that separate the molecules based on their charges. For a negatively-charged DNA molecule, it will migrate towards the positively-charged cathode. Electrophoresis gel is a matrix composed by cross-linked polymers from polysaccharides (agarose gel) or acrylamide (polyacrylamide) that form mesh networks for separating the nucleic acid molecules. By varying the concentration of agarose or polyacrylamide, the porosity of the gel is adjusted according to the molecular size of DNA molecules. When subjected to electric field, the DNA molecules will migrate through the matrix at different rates accounted by its molecular size and charge. Smaller molecules (E.g. DNA) have a greater migration distance than large molecules (E.g. RNA molecules).
- Density gradient agent: Glycerol or sucrose will increase the density of the DNA material so that it will be layered at the base of the agarose gel's sample well for separation.
- Tracking dye: Allow visualisation of the DNA sample and monitoring the progress of the electrophoresis.
- DNA ladder: Contains DNA molecules with different molecular sizes (in base pairs) to approximate the size of the unknown DNA molecules.
With the advancement of current technology, the genescanner has the ability to quantify the amplicons based on the fluorescent intensity. Gel electrophoresis is no longer used after PCR for quantification. The main use of gel electrophoresis is to identify any false postitives that may arise from non-complementary hybridization between the primers and DNA template strand.
3) Quantification by fluorescent intensity
Quantification may be performed during amplification process or gel electrophoresis. If PCR was performed in the spectrofluorometric thermal cycler, fluorescent intensity can be monitored at the annealing phase of the thermal cycle. Alternatively, if quantification occurs in gel electrophoresis, the DNA samples have to be substantially separated before excitation by irradiation for quantification. The fluorescent signal can be enhanced by a photomultiplier and subsequently analyzed by the computer. The fluorescent intensity is the difference in the initial fluorescence and level after the amplification. The gene scanner detects the level of fluorescence emitted from the fluorescent probes after cleavage by the Taq DNA polymerase in PCR. Fluorescence intensity is used to quantify the PCR products where the incremental amounts of DNA materials result in increased fluorescence.
- The principle of this quantification process arises from the irradiation (E.g. Laser) emitted from the genescanner. Depending on the fluorescent probe, the fluorescent dye will be absorbing light at a particular wavelength (E.g. MB-Green 485/530 nm, MB-Red 530/590 nm) and excited. Subsequent emission of fluorescence is derived from the dye through resonance energy transfer. This energy transfer will be increased by the distancing the fluorescent dye and the quencher group in the amplification. Quantity of the PCR products is determined by the relative intensities of fluorescence[9].
Comparison against Conventional PCR
Before we begin to compare fluorescent PCR (F-PCR) against conventional PCR (C-PCR), it is important to recall the laboratory procedures from F-PCR and C-PCR. In order to obtain substantial quantity and molecular size of genetic material, PCR cycles have to be completed. In C-PCR, agarose or acrylamide gels are separated by an electric field according to their molecular size. Smaller amplicons are migrating at a greater rate through the gel. However, C-PCR can be made more accurate by labelling primers with fluorescent markers that is currently known as F-PCR. A sensitive system fluorescent DNA sequencer, also known as a gene scanner can be used to separate and analyze the F-PCR products. therefore F-PCR allows detection of product without agarose or acrylamide gel electrophoresis.[10]
Advantages of Fluorescent-PCR
These are the advantages provided by F-PCR when compared to C-PCR [11]:
|Conventional PCR
|Fluorescent PCR
|Less sensitive
|More sensitive by about 1000-fold
|Lower accuracy
|High accuracy (97-98%) from 1-2 base pairs difference
|Number of PCR cycles
|More PCR cycles required
|Less PCR cycles required for the same level of detection
|Loading of product
|More loading of products
|Less loading of products; more efficient because of more repeated sampling
|Detection reagents
|Radioactive labelled nucleotides used
|Fluorescent-tagged nucleotides used; less toxic
|Quantitative analysis
|Gel electrophoresis
|Detection of fluorescence signal intensity
|Detection of multiple products
|Separation of products by molecular size in gel electrophoresis
|Different fluorescent dye to detect multiple products
Disadvantages of Fluorescent-PCR
The potential disadvantages that may arise from this technique include:
- Cross contamination can easily produce false positive results.
- Specificity: Amplified PCR products can only be obtained when conditions are specific.
- Slight differences can reduce yield dramatically.
- There are different requirements for each primer set; thus all conditions are optimized to maximize the yield of PCR products.
- Optimization of many procedural conditions is essential for multiple reaction where several different primer sets are used.
- Conditions will include temperatures for different phases of thermal cycle, magnesium concentration etc.
- The technique is costly and requires specialized equipments.
Clinical Applications of Fluorescent-PCR for Diagnosis
Identification of Retroviruses
A multiplex nucleic acid assay was developed by Vet et al., 1999, that both identified and quantified the abundance of retroviruses including the HIV-1, HIV-2 and human T-lymphotropic virus type I and II. Amplification of the retroviral DNA sequences was performed through PCR assays in spectrofluorometric thermal cycler. The amplified retroviral DNA were hybridized to specific fluorescent probes that includes fluorescein for HIV-1, tetracholoro-6-carboxyfluorescein (TET) for HIV-2, tetramethylrhodamine (TMR) for HTLV-I and carboxyrhodamine (RHD) for HTLV-II. The fluorescence colour would be crucial for identification of the specific retroviruses. Fluorescence spectrum at 500-650 nm was detected from the assay sample during the annealing phase of the thermal cycle. Quantification of the retro-viral DNA abundance was conducted in real-time, where the intensity of the fluorescence signal was increased significantly with the number of thermal cycles. The reliability of the assay was demonstrated with clinical samples. The retroviruses were identified and false positives were eliminated. Therefore, through the use of fluorescent-PCR in the assay, it has enhanced the efficiency and reliability of screening donated blood and transplanted tissue for retroviruses.
Results were plotted with fluorescence intensity of fluorescent probes against the number of completed thermal cycles. The colour of the fluorescence acted as the indicator of the of the retroviral DNA that was amplified. In the laboratory samples, the results have shown that there is a positive correlation between the fluorescence intensity and the number of thermal cycles. Increase in fluorescence intensity were observed for all four nucleotide sequences of HIV-1, HIV-2, HTLV-I and HTLV-II. Elevation of fluorescence signal resulted from the higher target copy number of the amplified retrovirus DNA.
In the clinical samples, the retroviruses were correctly identified by the assay indicated by the colour of the fluorescence. In samples of healthy individuals, the assay correctly gave a negative result due to the absence of retrovirus DNA. The PCR assays allow fast and sensitive detection of the amplified retrovirus DNA, which is essential for rapid screening of diagnostic samples. [12]
Analysis of Insulin-like Growth Factor Receptor 1 Gene Copy Number in Survival of Small-cell Lung Cancer
Quantitative fluorescent PCR technique has also been used in cancer research. One study focused into the correlation between that of two growth factors, insulin-like growth factor-1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), being expressed in non-small-cell lung cancers (NSCLC). IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction. Fluorescent in situ hybridization was subsequently used to assess IGF1R gene expression using customized probes. IGF1R and EGFR protein expression showed significant correlation and presence within cancers. Prognosis of the cancer is dependent on the gene copy number. Amplification of HER2 or high EGFR gene copy number is related to the malignancy and poor survival of patients. It was concluded that IGF1R gene expression does not associate with survival, whereas high IGF1R gene harbors positive prognostic value. By utilising fluorescent PCR, prognosis of patients can be determined by high gene copy number of HER2, EGFR and IGF1R.[13]
Performing Preimplantation Genetic Diagnosis for myotonic dystrophy or Steiner's disease
Myotonic dystrophy also known as Steiner's disease is described as a disease that causes muscular weakness, atrophy and myotonia. It is also an autosomal dominant disease. The disease affects the distal muscles of the limbs; frontal baldness; atrophy of the sternomastoids and gonads; lens opacity; mild endocrine and bone changes as wells as cardiomyopathy. In 1992 the gene on loci 19q was located and cloned and it was observed to contain CTG triplet repeat. Normal individuals were known to contain 5-30 repeats; mildly affected patients have 50-80 repeats; severely affected have over 200 repeats. This repeat was used to diagnose and develop a preimplantation genetic diagnosis (PGD) for myotonic dystrophy. When conventional-PCR (C-PCR) was used, it was decided that a setback of this assay was the loss of healthy embryos that were misdiagnosed as affected leading to a high allelic dropout (ADO) rate (more than 20%). In comparison,F-PCR ADO rate was found to be much lower because it is more sensitive to C-PCR. Therefore, their study concluded that F-PCR for PGD is superior to conventional PCR due to its superior accuracy leading to a lower ADO rate and subsequently there will be less embryos lost due to misdiagnosis.[14]
Detection of Mycobacterium tuberculosis Genome DNA
Fluorescence correlation has been utilized to detect specific specific in-vitro amplification of the genetic sequences. The fluorescent signal was detected by fluorescence correlation spectroscopy (FCS) after probe extension during PCR amplification of a specific sequence for Mycobacterium tuberculosis. The pathogenic genome from Mycobacterium tuberculosiswas detected after subjecting to PCR amplification with Stoffel fragment of Thermus aquaticus DNA polymerase. This amplification was initiated in the presence of rhodamine-labelled probe binding to the targeted DNA sequences between the amplification primers. In the presence of the template, the probe is necessary to the formation of specific amplicons. Without the template DNA strand, the primers can initiate non-specific amplification that consequently yield unspecific products during PCR products. This would not interfere with the diffusion time of the low concentration fluorescent probe.
Results have shown that the correlation between the fluorescence signal and diffusion time of the probe was increased progressively with increasing PCR thermal cycle number. This observation has demonstrated the specificity of the PCR amplification. Therefore, low input of genome could be quantified without the need of manipulation after PCR besides the reading from the FCS. However, it is necessary to avoid false-positives in diagnostic test by ensuring the targeting amplification is specific. Confirmation was accomplished by gel electrophoresis through processing the DNA sample in a gel. In this technique, low concentrations of probe were used. Therefore, it provides more stringent conditions for probe binding to its specific target. Thermus aquaticus DNA polymerase was used for DNA extension in this study. As compared to Taq DNA polymerase, Stoffel fragment of Thermus aquaticus DNA polymerase lacks the intrinsic exonuclease activity that explained the low concentration of probes utilized in the study.
Mycobacterium species are slow growing pathogens. Thus, identification of these pathogens is reliant on the long and laborious culturing techniques. The PCR technique has improvised the analysis techniques for diagnosis. Rapid diagnosis is critical for treatment and preventing the resurgence of tuberculosis worldwide [15].
Fluorescent-PCR Applications in Cell Biology Research
Evaluating the effects of kinase inhibitors on epithelial to mesenchymal transition
Epithelial to Mesenchymal Transition (EMT) is the cellular event, which contributes to organogenesis, cancer and organ fibrosis. This event is triggered by the Transforming Growth Factor. Kinases are implicated in the signaling pathways that induce mesenchymal state. Inhibitors of the kinases in EMT were evaluated in this study for the reversal of the induced mesenchymal state.
Quantitative or fluorescent PCR are used to quantify the total RNA isolated from the cells by the UV spectrophotometer. Results have demonstrated that the treatment with a TRF-βI inhibitor will reverse the PA-1 RNA levels in TGF-βI induced mesenchymal renal tubular cells to baseline levels present in epithelial cells.
In addition, the kinase inhibitors in mono-therapy or combined therapy were evaluated on the expression of several altered genes in EMT. These genetic expression were examined by the fluorescent-PCR. It began with the induction to mesenchymal state by the incubation with TGF-βI; then the cells were treated with the kinase inhibitors. RNA level of Ksp-cadherin, SM22 and MMP-9 were determined by the fluorescent-PCR. Significant differences were observed between the untreated cells and the cells treated with kinase inhibitors. RNA levels were drastically altered by the administration of kinase inhibitors. Kinase inhibitors exert their therapeutic effects by inducing the accumulation of transcripts specific to epithelial cells or certain additional specific transcripts such as Ksp-cadherin associated with reversal of EMT. Through the fluorescent-PCR technique, the kinase inhibitors could be evaluated on their efficacy in reversing EMT. This conclude that the combination of the small molecule therapy that target several kinases may be necessary to reverse the disease conditions[16].
Studying the modulation of E2-stimulated expression of ERα target genes by Keratin 18
The excessive activation of estrogen receptor (ERα) and over-expression of its co-activators are attributable to the oncogenesis in breast cancer. LRP 16 is acting both as an ERα target gene and co-activator; therefore, it is involved in the proliferation of MCF-7 breast cancer cells. Intermediate filament protein keratin 18 (K18) is a LRP16 interacting protein. Expression of K18 in MCF-7 cells has significantly lowered the association of LRP16 with ERα. Consequently, it attenuated ERα-activated reporter gene activity and decreasing gene expression by inhibiting ERα recruitment to the DNA.
To study if K18 affects E2 induction of ERα target genes in MCF-7 cells, fluorescent- or quantitative PCR was used to measure the mRNA expression levels of cyclin D1 that is known to be E-2 regulated in MCF-7 cells. Over-expression of the K18 has attenuated this induction. While the over-expression of LRP 16 has antagonize the K18 inhibition of E2-induced expression of target genes. The MC7 cells were cultured and transfected with the indicated vectors. The RNA were extracted from the cells for analysis. The expression of the indicated RNA abundance was analyzed by the fluorescent-PCR.
From the observations, it has been concluded that K18 has the role of sequestering LRP 16 in the cytoplasm. Therefore, it prevents the nuclear action of LRP 16 and attenuates ERα signalling. As a result, it will blunt estrogen-stimulated cell-cycle progression of ERα-positive breast cancer cells. The level of K18 gene can be used as a marker for the diagnosis of breast cancer; where the level of K18 gene expression correlates inversely with the progression of breast cancer[17].
- Amplicon: The amplified sequence of DNA in the PCR process.
- Atrophy: A wasting or decrease in size of a body organ, tissue, or part owing to disease, injury, or lack of use.
- Autosomal disease: A disease caused by a gene located on a chromosome other than a sex chromosome (autosomal chromosome).
- DNA duplex: Known as DNA double helix; it is a typical conformation of a double-stranded DNA molecule in which two polynucleotide strands are wounded around each other with base pairing between the double strands.
- DNA template strand: Also known as parenteral DNA strand is the DNA or RNA sequence that is going to be amplified.
- Dominant disease: A disease caused by an affected individual who has inherited one copy of a mutant gene and one normal gene on a pair of autosomal chromosomes. Individuals with autosomal dominant diseases have a 50-50 chance of passing the mutant gene and therefore the disorder onto each of their children.
- EDTA: Ethylenediaminetetraacetic acid; a complex molecule used to chelate magnesium ion (as a co-factor for DNA polymerase) that subsequently inhibits the action of DNA polymerase to extend the DNA strand.
- Fluorophore: The fluorescent dye used to monitor dye accumulation.
- Fluorescent probe: A probe which is labelled with a fluorescent dye, so that the signal emitted can be captured by photometric methods.
- Gene copy number: A copy number variation (CNV) is a segment of DNA in which copy-number differences have been found by comparison of two or more genomes.
- Hybridization: Experimental process in which two complementary nucleic acid strands form a double helix; a powerful technique for detecting specific nucleotide sequences.
- In-situ hybridization: Technique in which a single-stranded RNA or DNA probe is used to locate a gene or an mRNA molecule in an entire cell or tissue.
- Intrinsic nuclease activity: Property of the Taq polymerase to catalyze the hydrolysis of terminal nucleotide from the nucleic acid chains.
- in vitro: Term used by the biochemist to describe a process taking place in an isolated cell-free extract. Also used by cell biologists to refer to cells growing in culture (in vitro), as opposed to in an organism (in vivo).
- in vivo: In an intact cell or organism.
- Molecular beacons: These hairpin probes consist of a sequence-specific loop region flanked by two inverted repeats. Reporter and quencher dyes are attached to each end of the molecule.
- Myotonia: Tonic spasm or temporary rigidity of one or more muscles, often characteristic of various muscular disorders.
- Oligonucleotides: A short polymer composed of nucleosides with a series of one or more phosphate groups joined by an ester linkage to the sugar moiety.
- Organogenesis: The formation and development of the organs of living things.
- Plasmids: Small circular DNA molecule that replicates independently of the genome. Used extensively as a vector for DNA cloning.
- Primers: A short length of RNA made at the beginning of a DNA synthesis event catalyzed by DNA polymerase; these RNA primers are subsequently removed and filled in with DNA.
- Quencher: The molecule that absorbs the emission of fluorescent reporter dye when in close vicinity (6 to 10 nucleotides). It is also called as the acceptor dye.
- Restriction enzymes: Nuclease that can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs. Different restriction enzymes cut at different sequences.
- Resonance energy transfer: The interaction between the electronic and excited state of two dye molecules. The excitation is transferred from the donor dye molecule to the acceptor dye molecule.
- Radioactive-labelled nucleotides: A defined segment of nucleic acid that is tagged with a radioactive marker. It is necessary to identify the specific segments of DNA that carries the complementary sequence.
- Retrovirus: RNA-containing virus that replicates in a cell by first making a double-stranded DNA intermediate. This DNA is inserted into the cell's chromosome, where it can be maintained for a long time and is transcribed to produce new viral genomes and mRNAs that encode viral proteins.
- Stoffel fragment: An unique enzyme is a 61 kDa modified form of recombinant Taq DNA Polymerase from which the N-terminal 289 amino acids have been deleted to increase stringency at lower ionic strength and reduce misextension.
- Thermal cycling: A process of temperature modulation that consists of cycles of repeated heating and cooling of DNA for denaturation and enzymatic extension of the DNA template.
- Vectors: Genetic element, usually a bacteriophage or plasmid, that is used to carry a fragment of DNA into a recipient cell for the purpose of gene cloning.
- Wild-type: Normal, non mutant form of a species resulting from breeding under natural conditions.
- ↑ EJ Sambriskia,DC Schwartzc, JJ de Pabloa. Uncovering pathways in DNA oligonucleotide hybridization via transition state analysis. Proceedings of the National Academy of Sciences. 2009; 43: 18125-18130.Proceedings of the National Academy of Sciences (PNAS)
- ↑ LF Cavalieri, BH Rosenbery. Nucleic acids: Molecular biology of DNA.Annual Review of Biochemistry. 1962; 31: 247-270.
- ↑ Arnheim N, Erlich H. Polymerase Chain Reaction Strategy. Annu. Rev. Biochem. 1992. 61:131-156.
- ↑ Paul Rabinow. Introduction. Making PCR. The University of Chicago Press, 1996. pp: 1-19
- ↑ Hauge B, Oggero C, Nguyen N, Fu C, Dong F, 2009 Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression. PLoS ONE 4(9): e7205. doi:10.1371/journal.pone.0007205 Public Library of Science (PLoS One)
- ↑ Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999 August 3; 96(16): 9236–9241. PMCID: PMC17763 PNAS
- ↑ Morrison LE. Basic principles of fluorescence and energy transfer applied to real-time PCR. Mol Biotechnol. 2010 Feb;44(2):168-76. Review. PubMed PMID:19950004.PubMed
- ↑ Vet AM, Majithia AR, Marras AE, Tyagi S, Syamalima D, Poiesz BJ, Kramer FR. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl . Acad. Sci . USAVol. 96, pp. 6394 – 6399 Proceedings of the National Academy of Sciences (PNAS)
- ↑ I Findlay and P Quirke.Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells.Hum Reprod Update. 1996 Mar–Apr; 2(2): 137–152.PubMed
- ↑ A J Woolford and J W Dale. Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing.FEMS Microbiol Lett. 1992 December 1; 78(2-3): 311–316.PubMed
- ↑ I Findlay and P Quirke.Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells.Hum Reprod Update. 1996 Mar–Apr; 2(2): 137–152.PubMed
- ↑ Vet AM, Majithia AR, Marras AE, Tyagi S, Syamalima D, Poiesz BJ, Kramer FR. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl . Acad. Sci . USAVol. 96, pp. 6394 – 6399 Proceedings of the National Academy of Sciences (PNAS)
- ↑ Dziadziuszko R, Merrick DT, Witta SE, Mendoza AD, Szostakiewicz B, Szymanowska A, Rzyman W, Dziadziuszko K, Jassem J, Bunn PA Jr, Varella-Garcia M, Hirsch FR.Insulin-like growth factor receptor 1 (IGF1R) gene copy number is associated with survival in operable non-small-cell lung cancer: a comparison between IGF1R fluorescent in situ hybridization, protein expression, and mRNA expression.J Clin Oncol. 2010 May 1;28(13):2174-80.
- ↑ K Sermon, A De Vos, H Van de Velde, S Seneca, W Lissens, H Joris, M Vandervorst, A Van Steirteghem, and I Liebaers. Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert's disease). Mol Hum Reprod. 1998 August; 4(8): 791–796.
- ↑ NJ Walter, P Schwille, M Eigen. Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR.PNAS.1996; 93: 12805-12810.Proceedings of the National Academy of Sciences (PNAS)
- ↑ S Das, B N Becker, F M Hoffmann and J E Mertz1. Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway. BMC Cell Biology. 2009; 10: 94.BMC Cell Biology
- ↑ Y Meng, Z Wu, X Yin, Y Zhao, M Chen, Y Si, J Yang, X Fu, W Han. Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm. BMC Cell Biology. 2009; 10:96. doi:10.1186/1471-2121-10-96 BMC Cell Biology
2010 Projects
Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer


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A couple of new red-band clips for director Steven Soderbergh's Magic Mike have gone online. Though there aren't any gratuitous full-frontal shots, there's plenty of shirtlessness, pantslessness and ass-shots to put the rest of us members of the male species to shame. I'm beyond eager to see what Soderbergh has put together with this "stripper movie" and based on general buzz, the unofficial Year of Channing Tatum should continue full speed ahead on the heels of Magic Mike.
Hit the jump to watch the NSFW trailer and TV spot, and if you missed 53 high-resolution images from the film click here. The film also stars Matthew McConaughey, Alex Pettyfer, Matt Bomer, Joe Manganiello, Olivia Munn, Kevin Nash, Riley Keough, Cody Horn and Adam Rodriguez. The film opens on June 29th.
Trailer and TV Spot via Digital Spy.
Here's the synopsis for Magic Mike:
Mike (Tatum) is an entrepreneur. A man of many talents and loads of charm, he spends his days pursuing the American Dream from as many angles as he can handle: from roofing houses and detailing cars to designing furniture from his Tampa beach condo. But at night… he's just magic. The hot headliner in an all-male revue, Magic Mike has been rocking the stage at Club Xquisite for years with his original style and over-the-top dance moves. The more the ladies love him, the more they spend, and the happier that makes club owner Dallas (McConaughey).
Seeing potential in a guy he calls the Kid (Pettyfer), Mike takes the 19-year-old under his wing and schools him in the fine arts of dancing, partying, picking up women and making easy money. It's not long before the club's newest act has fans of his own, as the summer opens up to a world of fun, friendship and good times. Meanwhile, Mike meets the Kid's captivating sister, Brooke (Horn). She's definitely someone he'd like to know a lot better, and it looks like he has a chance…until his lifestyle gets in the way.
Directed by Oscar winner Steven Soderbergh ("Traffic") and written by Reid Carolin, "Magic Mike" is produced by Nick Wechsler, Gregory Jacobs, Channing Tatum and Reid Carolin. The creative filmmaking team includes production designer Howard Cummings, costume designer Christopher Peterson, music supervisor Frankie Pine and choreographer Alison Faulk.
This film is rated R by the MPAA for pervasive sexual content, brief graphic nudity, language and some drug use.


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A prisoner has a limited supply of paper and ink so to save resources, he decided to refrain from using letters that extend above or below the line (b, d, f, g, h, j, k, l, p, q, t, or y). With this kind of constraint, can he write anything worthwhile?
Well, depending on the prisoner's level of creativity, a lot can actually be written. Ian Monk offered the following:
a russian con's economic missive
we were once seven con men, we are now seven cons. as communism was over we saw easier success in american consumerism, i.e. crime. in a moscow inn, we swore: — seven is one, so one is seven … soon we came across a scam. our main man wove us a nice wee earner: — we own a zinc mine. since our russian economic crisis came in, our income's never risen. we can cram ice in our mine's veins, raise rumours re our ice mine's immense resources, con morons we are mere zeros. as soon as career men see our ice, we win 'em over. once we've won 'em over, we receive numerous ecus or euros. as soon as we've our monies, we serve 'em arsenic in wine. we can even recommence on numerous occasions. … our scam was a success. our asses never saw sense. we were euros in. we saw our main man serve our vicious wine mix … a near miss .. our arsenic was mere mouse venom. some asses were survivors: — summon a coroner, someone swore. — or a nurse. — or some rozzers. so we ran. we swam across a river. as soon as no one was near us, we wove our monies in wee canvas cases we wore in our arses. we ran on. in vain … someone saw us on vanavara's main avenue. a commissioner, nine rozzers, seven airmen, six cia men overcame us. we were sworn in. we are now in moscow in irons in room nine. as soon as someone receives or sos, come … run … save us … since no one's ever come across our economies, our ransoms are even now in our arses.
Reference (Click to Show)
Harry Matthews et al (2005), Oulipo Compendium