MLPA
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MLPA (Multiplex Ligation-dependent Probe Amplification) is a technique that allows for the relative quantification of up to 45 genomic DNA sequences in one reaction using PCR. It requires only a small amount of DNA and can detect changes in copy number that differ by a single nucleotide. The MLPA technique involves denaturation of DNA samples, hybridization of probes, ligation of probes, amplification by PCR, and analysis via capillary electrophoresis. MLPA kits are available for detection of aneuploidies, deletions/duplications associated with genetic disorders, and gene expression changes in cancer.
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PCR
This document provides an overview of polymerase chain reaction (PCR) including its history, principles, components, cycle, limitations, advantages, disadvantages, types, and applications. PCR is described as an in vitro method for enzymatically synthesizing specific DNA sequences using two oligonucleotide primers that flank the target region. It allows for exponential amplification of minute amounts of DNA. Real-time PCR is highlighted as an advancement that provides ease of quantification, greater sensitivity, reproducibility, and precision compared to traditional PCR. The document also reviews various applications of PCR in fields such as medicine, forensics, and research.
Serological methods.pptx
1. Serological techniques are important tools for detecting and diagnosing plant pathogenic bacteria. These techniques rely on the reaction between antigens and antibodies.
2. Several serological assay techniques can be used, including PCR-based methods, LAMP, microarrays, agglutination tests, and the Ouchterlony double diffusion test.
3. PCR is used to amplify target nucleic acid sequences and was invented by Kary Mullis. It involves denaturation, annealing of primers, and extension of the DNA strands. Variations include RT-PCR, nested PCR, and multiplex PCR.
Isothermal Nucleic Acid Amplification Techniques
This document provides an overview of various isothermal nucleic acid amplification techniques, including:
1. TMA, NASBA, SMART, SDA, RCA, LAMP, and others that amplify nucleic acids under isothermal conditions to overcome limitations of PCR like high costs and contamination risks.
2. It describes several techniques in detail, explaining their mechanisms which involve primers, polymerases, and isothermal reactions to exponentially amplify target sequences.
3. The techniques have applications in research, medicine, forensics and more due to their simplicity, lower costs, and ability to be used at point-of-care without specialized equipment.
2013 polymerase chain-reaction-shakira sulehri
The document discusses polymerase chain reaction (PCR). It describes PCR as a technique that amplifies specific DNA sequences using DNA polymerase. The key components of a PCR reaction are a template DNA, primers, DNA polymerase, nucleotides, and buffer. Through repeated heating and cooling cycles, the target DNA is amplified exponentially. The document outlines the three phases of PCR - exponential, linear, and plateau. It also discusses various types of PCR like real-time PCR, nested PCR, and their applications in fields like genetics, forensics, and disease diagnosis.
POLYMERASE CHAIN REACTION (PCR) AND ENZYME-LINKED.pptx
Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are molecular biology techniques. PCR is used to amplify small amounts of DNA and was developed by Kary Mullis, who won a Nobel Prize for his work. ELISA is a common immunological assay that uses antibodies and enzymes to detect antigens or antibodies in a biological sample, allowing quantification of proteins. Both PCR and ELISA have wide applications in biomedical research, forensics, disease diagnosis, and more.
Tools used in molecular biology
This document provides information about genetic polymorphisms and methods used to detect single nucleotide polymorphisms (SNPs), including DNA isolation, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and gel electrophoresis. It defines polymorphisms and SNPs, and describes how restriction enzymes can be used to detect SNPs by cleaving DNA into different fragments depending on nucleotide variations. The document outlines the steps of PCR including melting, annealing, and extension cycles to amplify DNA, as well as how to perform restriction digests and electrophoresis to separate DNA fragments by size for analysis.
Tools used in molecular biology
This document provides information about genetic polymorphisms and methods used to detect single nucleotide polymorphisms (SNPs), including DNA isolation, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and gel electrophoresis. It defines a polymorphism as a harmless DNA variation between individuals and SNPs as single base mutations that are the most common source of genetic variation. The document outlines the steps of PCR including melting, annealing, and extension cycles to amplify DNA, as well as how restriction enzymes are used to digest PCR products into different fragments depending on sequences, which can then be separated by size via gel electrophoresis to detect SNPs.
4. Brief introduction to Polymerase Chain Reaction.pptx
The document provides an introduction to polymerase chain reaction (PCR), which is a technique for amplifying specific DNA sequences. PCR allows for generating billions of copies of a target DNA fragment. It involves repeated cycles of denaturation, annealing of primers to the DNA templates, and extension of the primers by DNA polymerase. This process amplifies the target DNA exponentially. Key requirements for PCR are a DNA template, primers, DNA polymerase, and nucleotides. Automated PCR machines allow for rapid and precise amplification through repeated temperature changes. PCR has many applications in medicine, infectious disease detection, forensics, and molecular genetics research.
Overview of the glossary related to pcr
This document provides definitions and explanations of key terms related to polymerase chain reaction (PCR). It defines PCR as a method used to exponentially amplify specific DNA sequences. It then explains several essential PCR concepts in 3 sentences or less, including absolute quantification, template, threshold, amplicon, anchor and reporter probes, fluorescence resonance energy transfer, baseline value, end-point analysis, calibrator, Ct value, internal positive control, melting curve analysis, nucleic acid sequence-based amplification, non-template controls, nucleic acid target, quencher, reporter dye, relative quantification, and TaqMan probe.
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MLPA
- 1. MLPA ® Multiplex Ligation Probe Amplification MRC-Holland b.v.
- 10. Separation and quantification by capillary electrophoresis Each peak is the amplification product of a specific probe. Samples are compared to a control sample. A difference in relative peak height or peak area indicates a copy number change of the probe target sequence
- 11. Detection of Chr X copy number X Triple X Female Male 283 bp 346 bp
- 12. Specificity of MLPA probes is very high. control Homozygous deletion ASPA exon 1-6 1 5 4 3 2 6 1 5 4 3 2 6
- 13. Reproducibility of MLPA is sufficient to distinguish homozygotes and heterozygotes. Heterozygous ASPA del. exon 1-6 Control 1 5 4 3 2 6 1 5 4 3 2 6
- 14. 55 o C hybridisation 60 o C hybridisation 62 o C hybridisation Many variables have only a small influence on MLPA results
- 15. 10 ng. DNA (less than recommended minimum amount) 50 ng. DNA 750 ng. DNA Amount of DNA used has little influence on MLPA results
- 17. Normal Ligation of the two probe oligonucleotides Amplification product Mismatch at the probe ligation site No ligation, no amplification product MLPA discriminates sequences that differ in only a single nucleotide and can be used to detect known mutations. Mismatch Perfect match
- 18. MLPA protocol The use of a thermocycler with heated lid is essential. 1. Denature 20-500 ng DNA by heating to 98 o C. 2. Add the MLPA probes and MLPA Buffer. Incubate o/n at 60 o C. 3. Add Ligase-65 and ligase buffer. Ligate at 54 o C for 15 minutes. 4. Inactivate the ligase by heating to 98 o C. Add PCR primers, dNTPs and polymerase and start the PCR reaction. 5. Analyse the products by electrophoresis. 6. Export fragment length’s and peak areas to Excel
- 20. MS-MLPA Only undigested (methylated) and ligated probes are exponentially amplified Unmethylated Target M M Methylated Target Denaturation and Multiplex probe hybridization M Ligation and Digestion with methylation sensitive endonucleases M
- 21. Control Undigested ME028 PW / Angelman kit Control Hha1 digested Arrows indicate probes for imprinted sequences (1 copy methylated)