New PCR Machine Microchip

Researchers in Hong Kong have miniaturized technology needed to perform the versatile polymerase chain reaction (PCR) -- widely used in criminal investigations, disease diagnosis, and a range of other key applications. They report development of a long-sought PCR microchip that could permit use of PCR at crime scenes, in doctors' offices, and other out-of-lab locations.

I-Ming Hsing and colleagues note that PCR works like a biological copy machine, transforming a few wisps of DNA into billions of copies. However, existing PCR machines are so big and complex that they can be used only in laboratories. Scientists have searched for years for a portable, PCR technique that can be used outside the lab.

The study describes a new PCR technique that uses electrochemical DNA sensors to provide simultaneous DNA amplification and detection on a silicon-glass microchip. Their performance tests show that the new technique, called electrochemical real-time PCR (ERT-PCR), is about as fast and sensitive as conventional PCR. The new technique shows "tremendous" promise as a portable system for moving DNA analysis out of the lab and into remote locations, the researchers say.

The article "Electrochemistry-Based Real-Time PCR on a Microchip" is published in the Jan. 15 issue of ACS' Analytical Chemistry.

taken from : sciencedaily Jan 4th, 2008

monoclonal antibody industry...next bom industry ..???

Monoclonal antibody drugs offer several advantages over other types of drugs. Technologically, antibodies exhibit amazing specificity that allows for a safe and targeted attack on disease-causing cells or compounds. The antibody itself may be used as a weapon or the antibody may serve as the vehicle to deliver a drug. Along with high specificity, monoclonal antibodies are effective in a broad range of diseases including autoimmune, cardiovascular and infectious diseases, cancer and inflammation. Also of great importance is that antibodies are virtually never toxic.

As of May 2005, there were 18 therapeutic monoclonal antibody products on the U.S. market. Worldwide, there were an estimated 500 monoclonal antibody products in development by more than 200 companies for the treatment of virtually every debilitating disease. Approximately 80 of these were in clinical trials.

This BCC report provides detailed market analyses and industry trends. It quantifies and qualifies the emerging market for monoclonal and polyclonal antibody drugs and imaging products. Forecasts and trends are gleaned from industry sources as well as from considered assessments of available and emerging technologies. The study also examines strategies used by biotechnology companies and pharmaceutical firms to develop and market products in this explosive market sector.

We will be waiting for the next big industry .....!!!!!

Detection fragile X Using a little split out ?

Hello reader,

I just learning about the article about detection of Fragile X syndrome patients using methylation-specific PCR of the FMR1 gene. It has been reported that using this method could detect fragile X patient, especally in man with 100 % accuracy. I also have friend of mine in lab who did many samples with this method. All the samples were blood that was drawn from the samples. I am just wondering...We can develop the test like this USING a little split out..since it can be run in PCR......So in the future we can screen the people in large population, more cheapest than Southern, and in one single day....!!!!.....Hmmmmm...very interesting....!!!
-------------------------------------

here is the article :

INTRODUCTION

The fragile X syndrome (FRAXA) is the most common inherited form of mental retardation in man. The molecular pathogenesis of the disease generally involves expansion of a (CGG)_n trinucleotide repeat located on the 5' region of the FMR1 gene, leading to hypermethylation of the promoter and shutdown of gene expression (reviewed by Nelson, 1998; Kaufmann and Reiss, 1999). The molecular diagnosis of FRAXA has depended on simultaneous Southern blot analysis of (CGG)_n length and methylation status. However, this is a complex procedure and, to screen for FRAXA among mentally retarded children, we need simpler and cheaper PCR-based diagnostic tests.

The first PCR-based test that we developed was based on direct PCR amplification of the (CGG)_n trinucleotide repeat with primers flanking the microsatellite, with a product of 557 bp for the (CGG)₂₉ allele (Haddad et al., 1996). Conditions were established so that full mutations failed to amplify. To produce an internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 base pairs upstream the polymorphic region. In blind trials the PCR-based test showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. The test had two main disadvantages. Firstly, a normal (CGG)_n allele was preferentially amplified by PCR due to its smaller size and thus the PCR technique could not be used for the diagnosis of FRAXA in females, because they are heterozygous and would be scored as normal. For the same reason, mosaic patients with a normal sized allele might yield a false negative result. That is why the test was not 100% sensitive. The second drawback resulted from the "failure-to-amplify" characteristic of the test that thus could not provide a definitive diagnosis of fragile X syndrome. Although not quite suitable for medical diagnosis, the PCR test proved to be a useful tool for fragile X syndrome screening in populations of mentally retarded males (Haddad et al., 1999).

Recently Herman et al. (1996) developed an elegant PCR assay for methylation status of CpG islands. DNA samples are first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We wish to report the successful application of this methylation-specific PCR (MSP) for the study of the FMR1 promoter. This led to a much-improved method for PCR diagnosis of the fragile X syndrome in affected males.

MATERIAL AND METHODS

Patients

We used DNA from eight patients with fragile X syndrome, all confirmed by Southern blot analysis: five of these were ascertained in a screening study of mentally retarded boys in Brazil (Haddad et al., 1999), two were patients diagnosed at GENE and the other (NA06852) was obtained from the Coriell Mutant Cell Repository (Camden, NJ, USA). DNA samples from 42 normal controls were obtained from paternity testing cases at GENE.

PCR primers

For development of primers we used the data of Stöger et al. (1997) who described the pattern of cytosine methylation at the CpG island of the FMR1 gene. We designed two primer pairs: the first, 5'-AAATGGGCGTTTTGGTTTTCGC-3' and 5'-GCCAAAAATCATCGCGCATACG-3', produces a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other, 5'-TGTTTTTTATTAAGTTTGTGTAT-3' and 5'-ACCAAAAATCATCACACATACA-3', generates an 84-bp product from the treated non-methylated promoter. The strategy behind the primer development is shown schematically in Figure 1.

Methylation-specific PCR

DNA samples were treated with sodium bisulfite as described by Herman et al. (1996). They were then submitted to PCR amplification in separate tubes with primers specific for the methylated (M) or non-methylated (N) versions of the CpG island of the FMR1 gene. In the reaction with the M primers we also included primers specific for the methylated version of the SNRPN gene (Kubota et al., 1997). PCR was performed in a final volume of 13 µl using 0.65 µ AmpliTaq Gold (Perkin Elmer, Foster City, CA, USA) in the manufacturer's recommended buffer, 200 µM of each dNTP, 0.4 µM of each primer and 100 ng of human genomic DNA. Thermal cycling conditions were: initial denaturation at 95^oC for 5 min, followed by 35 cycles of 1 min of annealing at 53^oC for the M reaction and 43^oC for the N reaction, 1 min of extension at 72^oC and denaturation at 95^oC for 1 min. Afterwards, the PCR reaction products were separated by electrophoresis in a 6% polyacrylamide gel and visualized by silver staining.

RESULTS AND DISCUSSION

In normal males only the 84-bp fragment was seen (Figure 2), while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with visualization of a much weaker 84-bp band (Figure 2). The probable reasons that the 84-bp product did not disappear as could be expected are that methylation is generally not complete (Stöger et al., 1997) and that somatic mosaicism occurs in the length of the (CGG)_n repeat in complete mutations. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15 (Kubota et al., 1997) generating a fragment of 174 bp (Figure 2).

Using the methylation-specific PCR we identified with 100% specificity, sensitivity and accuracy, eight previously diagnosed FRAXA male patients mixed with 42 normal controls. In theory the test should not be prone to producing false positive results and should also be very sensitive, permitting diagnosis even in mosaics with normal-sized alleles. Indeed, we have found that we can still obtain a clear methylated product even when DNA from FRAXA patients is diluted 20-fold with normal male DNA. If needed, sensitivity could be further increased by the use of fluorescently labeled primers and detection in an automatic DNA sequencer.

Apparently the pattern of methylation in the promoter region of the FMR1 is identical in full mutations of FRAXA and in X inactivation in normal females (Stöger et al., 1997). Thus, the MSP test cannot be used to diagnose the fragile X syndrome in affected females, since they already have, in virtue of X inactivation, a methylated FMR1 promoter region.

In summary, methylation-specific PCR emerges as a simple and efficient method for assessing methylation in the FMR1 CpG island. Indeed, it may become the method of choice for diagnosis of the fragile X syndrome in mentally retarded males.

New finding on Fragile X Syndrome gene

Scientists at The Scripps Research Institute have discovered a new gene involved in fragile X syndrome, a condition that often shares many symptoms of autism. The discovery may lead to new tests or treatments for several neurological disorders.

The new gene has been dubbed FMR4. "FMR4 is a novel gene that is located in the same chromosomal neighborhood as FMR1, a well established causative gene in fragile X syndrome," said Claes Wahlestedt, a professor at the Scripps Research campus in Jupiter, Florida. "Like FMR1, FMR4 is silenced in fragile X patients and up-regulated in FXTAS (fragile X-associated tremor/ataxia syndrome), a disease that resembles Parkinson's disease. Our discovery could lead to the development of new diagnostic tests or even to novel therapies for these defects."

Fragile X syndrome affects thousands of patients worldwide with severe learning disabilities, often accompanied by anxiety disorders, obsessive-compulsive behavior, and attention deficit hyperactivity disorder. There are currently no therapeutic treatments available for fragile X syndrome. Approximately one-third of all children diagnosed with fragile X syndrome also have some degree of autism, according to The National Fragile X Foundation, including such behaviors as social anxiety, poor eye contact, and hand biting.

More than 16 years ago, scientists linked fragile X syndrome to inactivation of FMR1 gene expression, leading to the lack of a protein known as the fragile X mental retardation protein, now considered to be critical for neuronal function. Until the current study, no other functional gene other than FMR1 had been shown to be inactivated in the disorder.

However, Wahlestedt knew the FMR1 gene locus-a specific point on a chromosome-was not well mapped. Wahlestedt and his colleagues hypothesized that unknown regulatory genes might be transcribed from the region.

The new study shows at least one other functional gene-FMR4-from this genetic region is linked to fragile X syndrome, although the gene's exact role in the intact brain remains uncharacterized..

"FMR4 is the new neighbor on the block and should not be ignored," Wahlestedt said. "While there is no direct relationship between these two genes [FMR1 and FMR4] that we know of, our study shows that FMR4 is not a conventional gene-it's a non-coding RNA transcript. It's not a dead piece of the genome, it has a pronounced functional effect in human cultured cells.."

The Role of Non-coding RNA

Non-coding RNA (ncRNA) transcripts or genes produce functional RNA molecules (ncRNAs) rather than encoding proteins. These ncRNAs are active in a number of different processes, including RNA modification, chromosome replication, and protein degradation.

A number of studies have suggested that at least 40 to 50 percent of the mammalian genome becomes transcribed, Wahlestedt pointed out, but only one to two percent of these transcripts are translated into proteins. "Several studies suggest that some ncRNA genes can be involved in various human diseases," he said. "FMR4 certainly falls into that category."

According to the study, FMR4 directly affects human cell proliferation in vitro-when the gene is silenced, changes in the cell cycle and a rise in apoptosis or programmed cell death occur. Overexpression, on the other hand, leads to increased cell proliferation.

The full meaning of this anti-apoptosis function is still unclear. "It could be critical for some cells to live or die at a certain stage in development, but we don't know what cells those might be," Wahlestedt added. "The fact that FMR4 is widely expressed in the human brain in both embryos and adults may possibly indicate a broad function."

The new study underscores the growing awareness among scientists of the complexity and unpredictability of the human genome.

"We know now that our genome is very busy and very complicated," Wahlestedt said "A great deal of this newly found complexity is about the regulation of other genes. As evolution has progressed, particularly in the higher organisms, there has been a corresponding increase in the need for regulatory mechanisms-to maintain more control over genome. Non-coding RNAs are at the center of these regulatory mechanisms."

The FMR4 discovery also highlights the mission of The Translational Research Institute at Scripps Florida, which is focused on translating basic research like the discovery of FMR4 into potential new therapeutics. The Translational Research Institute has a structure similar to a drug discovery company, and many of the researchers have pharmaceutical experience.

In addition to Wahlestedt, other authors of the study include Ahmad M. Khalil, Mohammad Ali Faghihi, Farzaneh Modarresi, and Shaun P. Brothers of The Scripps Research Institute in Jupiter, Florida.

The study, A Novel RNA Transcript with Antiapoptotic Function is Silenced in Fragile X Syndrome, was supported by Conquer Fragile X Foundation (now part of National Fragile X Foundation) and The Scripps Research Institute Florida. Upon publication, the study will be available at http://www.plosone.org/doi/pone.0001486.

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One single rogue trader loses 7.2 Billion for Societe General, one of France\'s largest banks.

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=======

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Indirect immuno-incubation of cryo sections and cultured cells

This method is working well in my experience and gave good visualization as well. Enjoy it....

Cryosections;

1. Cut 5-7 µm cryosections and put them on Menzel Superfrost slides.
2. Immediately airdry or store at –80C .

Cultured cells;

1. Culture cells on coated (for example poly-l-lysine) glass cover slips.

3. Fix wit 3%PF for 10 min.

4. Permeabilize with methanol for 20 min.

5. Rinse in PBS- (PBS 1x) for 2 min.

6. Block for endogenous peroxidase during 30 min. at RT. in PBS/H₂O₂/azide

100 ml PBS 1x (0.1M PBS)

2 ml H₂O₂ 30%

1 ml sodiumazide 12.5% (toxic!!)

7. Rinse in PBS- for 2 min.

8. Rinse in PBS+ (PBS 1x/ 0.5% protifar/ 0.15% glycine), 2 times 2 min.

1L PBS, 5g protifar and 1,5g glycine.

9. Incubation with the primary antibody for 90 min. at RT, (or overnight at 4C). (100µl/slide)

10. Rinse in PBS+ 3 times 5 min.

11. Incubation with the peroxidase conjugated secondary antibody for 60 min. at RT.

12. Rinse in PBS+ 3 times 5 min.

13. Rinse in PBS- for 2 min.

14. Incubation with DAB-substrate (DAKO Liquid DAB substrate-chromogen system)

Use 20µl DAB solution in 1 ml Dako buffer, 100µl/slide during 4-8 min.

! Wear gloves; DAB is carcinogenic!

15. Remove DAB-solution by placing the slides in aqua dest.

16. Refresh aqua dest immediately.

17. Counterstain with haematoxylin for 5 min.

18. Rinse in tapwater for 10 min.

Cryosections;

19. Dehydration; alcohol 96% 1 min.

alcohol 100% 2x1min.

xylene 2x2min.

20. Mount with Entellan.

21. Dry overnight at 37°C.

Cultered cells;

19. Rinse with aqua dest.

20. Mount with aquamount.

21. Dry overnight at 37°C.

! Note that all dilutions are made in PBS +

DNA ISOLATION MINI

This methode can be performed well in your lab. Enjoy it...

1. Add 1 bacterial-colony from a plate to a culture tube with 5-10ml medium with antibiotics and shake ON at 37°C.

2. Save 1ml of bacterial-culture in tubes and store in 4°C.

3. Prepare bacterial pellet by centrifuging 10min; 3000rpm on RT

4. Resuspend in 200ml 1x Maxi-prep

5. Add 400ml 0.2M NaOH/1%SDS and shake

(400ml 5M NaOH/500ml 20%SDS in 10ml)

6. Leave 5-10 min on RT

7. Add 200ml 3M NaAc pH4.8 and mix

8. Spin directly for 10min on RT

9. Discard supernatant in new tube

10. Add 0.6xVolume of isopropanol to the supernatant and mix

11. Spin directly for 3min on RT

12. Take of the supernatant, dry pellet and dissolve pellet in 200µl TE or dH₂0 with 1mg RNase (stock 10mg/ml) and leave 30min on 37°C

13. Add 200ml phenol/chloroform, mix gently and spin 5min; 13krpm on RT

14. Take of the water phase and add 1/10Volume 2M NaAc and 0.6xVolume isopropanol (You also can use 2xVolume ice cold 100% EtOH, mix and store for a while in -20°C)

15. Centrifuge 15min; max speed on RT

16. Wash pellet with 70%EtOH

17. Centrifuge 5min; max speed on RT

18. Dry pellet and resuspend in TE or dH₂0