Commonly used molecular biology experimental techniques:
Centrifuge technology:
Is separated, enzymes, nucleic acids (DNA, RNA), one of the cells of the most common method for protein purification.
Electrophoresis (electrophoresis): charged particles in an electric field toward the electrode of opposite direction thereto electric charge movement phenomenon.
It can be used to separate different molecular weight biomolecules.
1. Protein Electrophoresis:
Uses: Quantitative protein.
2. The nucleic acid electrophoresis:
Use: isolation, identification, purification of nucleic acid recovered.
For example: I just need a length of about 300bp molecules. Then, after electrophoresis, the gel cutting process, the molecule can be cut only at 300bp.
Protein research related technologies:
1. Determination:
2. Structure Determination:
Determination of (1) a structure: find out the order of amino acids in a protein peptide chain.
Method: Edman degradation, mass spectrometry (MS, proteolytic, polypeptide chains into smaller segments detected peptides.)
(2) the spatial structure determination: Protein Structure Analysis of Spatial Analysis is much more complicated than the primary structure.
Method: X-ray crystallography, nuclear magnetic resonance and the like.
3. Functional assay:
(1) Yeast two-hybrid (YTH):
Assumptions: To detect the interaction of protein X and protein Y.
Detection method:
The X and BD protein reporter gene fusion transcription factor;
AD fusion proteins with Y;
Complex protein X and protein Y to confirm the formation of activated gene expression can report. If the expression of the reporter gene can be activated, Note: X and Y form a complex, the close BD and AD, activated downstream reporter gene expression; the other hand, the reporter gene is not expressed.
principle:
Eukaryotic transcription factors (especially the GAL4 transcription factor of yeast), comprises two separated from one another, but the essential function domains: a binding domain with the DNA -BD; is a transcriptional activation domain -AD. Effect of the gene upstream sequence elements to identify the transcription and bind BD; the AD action by the other components of the transcription complex, downstream of the promoter gene transcription.
Even if separated from BD and AD, but if more spatial proximity can activate transcription. - the BD transcription factors, the AD this feature, whether to activate the transcription factor by detecting the expression of the effector gene can investigate whether protein X and Y interact.
(2) Protein chip technology: a high-throughput miniaturized automated protein analysis techniques. One test can detect hundreds or even thousands of target protein or polypeptide.
(3) Western blot blotting analysis by Western : Principle of using antigen-antibody reaction specific.
Uses: detecting whether there is a specific protein in a sample as well as semi-quantitative analysis, the study of protein interactions.
(4) immune coprecipitation given technology (co-immunoprecipitation, Co-IP )
(5) GST pull-down technique
(6) Bioinformatics prediction of protein
Nucleic acid hybridization (nucleic acid hybridization): refers to different sources but denatured nucleic acid molecule having a certain homology, under certain conditions refolding pairing between single-stranded each other to form a heteroduplex process.
In practice, use is often labeled a known sequence of a specific nucleotide fragment (i.e., the nucleic acid probe ) were hybridized with the sample to be tested, to determine the presence or absence of a particular nucleic acid sequence.
Nucleic acid probe (Probe), according to their nature and origin, genomic DNA probes, cDNA probes, RNA probes, and artificial synthetic oligonucleotide probes.
(1) Genomic DNA probe: preparing simple, easily degradable, maturity labeling methods, it is the most commonly used probes.
(2) cDNA probe: DNA as a template mRNA molecule synthesized under the catalysis of reverse transcriptase.
cDNA probes are coding sequences, introns and highly repetitive sequence is not present, the difference in the expression of certain genes probed more aspects of the application.
(. 3) RNA probe: was a labeled RNA molecule complementary thereto for detecting mRNA or DNA strand.
RNA probes hybridized with obvious advantages in terms of efficiency, pros and cons to observe gene transcription status, antisense nucleic acid research.
(4) synthetic oligonucleotide probes: If only the amino acid sequence of the protein alignment, without knowing the base sequence of the gene encoding may be utilized synthetic oligonucleotide probes to probe the unknown sequence of the gene.
The type of nucleic acid hybridization:
(1) Southern blot hybridization: The test electrophoretic separation DNA fragments were transferred and bonded to certain solid phase support, and a method for detecting hybridization with labeled DNA probes.
(2) Northern blot hybridization: one kind of the RNA blot test sample.
(3) In situ hybridization (ISH): does not change the position where the nucleic acid, hybridization with the probe directly. It can be divided situ hybridization and colony hybridization.
In situ hybridization: a) fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) - can be distributed precisely locate the gene on the chromosome; may be tissue or cell specific spatial distribution or RNA expression levels were determined and . Resolution up to 100-200Kkb. b) Genomic in situ hybridization (genomein situ hybridization, GISH): mainly used for comparative genomics. Using the difference in homology between species genomic DNA, the genomic DNA of a species as a probe, in situ hybridization with chromosomes of another species, the distribution and the degree of strength analysis of hybridization signals on chromosome, identification of different species comparison genetic relationship between the genome to explore the mechanism and rate of evolution of the genome.
(4) dot blot hybridization and slot blot hybridization: The site directly after denaturation of DNA or RNA sample to a solid support membrane, UV cross-linking or baking was post-fixed with a nucleic acid probe molecules hybridize to a method of detecting, The whole process does not require electrophoresis.
Several advantages compared to before: simple and quick operation, a plurality of film samples can be detected. Disadvantages: nucleic acid sample can not be detected by molecular weight measurement.
Uses: detecting copy number changes, the level of gene transcription.
Microarray (gene chip):
Comprising a DNA chip or DNA microarray, cDNA chips, a method for high-throughput detection of gene expression to establish based dot blot, it a number of known sequence of the oligonucleotide probe or cDNA to a solid surface as a solid probe, and then hybridized with labeled sample nucleic acid, by detecting a hybridization signal analysis, to obtain the various sequences of nucleic acids and the expression of the test information.
Features: High-throughput, large-scale, high sensitivity, high automation.
Uses: gene expression profiling, comparative research and discovery of new genes, DNA sequence analysis, gene diagnosis, drug screening, drug discovery, aspects of rational drug use, identification of herbs genome.
RNA expression in the cells can be detected, changes in copy number of the genome of a cell, SNP genome.
PCR (polymerase chain reaction, PCR) are mediated by a primer (Primer), a method using a DNA polymerase in vitro amplification of the gene, also known as gene amplification.
1. Reaction system: a template DNA, a thermostable DNA polymerase, two primers, four kinds of dNTP, Mg2 + containing buffer.
2. The course of the reaction: generally composed of 25 to 35 cycles, each cycle comprising three reaction steps.
(1) degeneration. The reaction was heated to 94 to 95 degrees, about 30 seconds, so to be amplified into a double stranded DNA were completely melted, the polymerization reaction as a template. If long or high GC content DNA fragment is necessary to set a higher temperature and longer time to assure complete solution of the template strand.
(2) annealing. The temperature was rapidly lowered to a suitable temperature and for 30 seconds to 3 'end of the primer to the template DNA strand of two complementary mating. Due to the short primer fragments, the structure is simple, and the number of far more than the number of template DNA, so the opportunity to binding between single-stranded DNA template minimal.
(3) extend. The reaction system temperature was raised to 72 ° C, then DNA polymerase will be single-stranded DNA as template, single nucleotide added individually to the 3 'end of the primer, so that the new chain extended continuously, until the end of the synthesis.
3. Detection of PCR products: After completion of the PCR analysis of the need for rigorous testing to determine whether to give the desired product. Common method of detection is as follows:
Gel electrophoresis, zymography, sequencing analysis, nucleic acid hybridization.
4.PCR purified product:
: Available phenol - chloroform extraction Tigard ethanol precipitation, electrophoresis may be directly target band was excised, and then using commercial gel extraction kit and purified.
PCR primers:
(1) Length: generally 15 ~ 30nt.
(2) 3 'terminus of the primer: This is where the extension begins, must not mismatch occurs, can not be modified.
5 'end (3) primer: 5'end of the primer may be a degree of modification, such as adding restriction sites, labeled biotin, fluorescein, digoxigenin, a primer mutation sites or mutant sequence, introducing the promoter sequence Wait.
(4) There are other features, such as: base composition, primer primer concentration own structure.
Applications of PCR technology: obtaining a target gene, site-directed mutagenesis, gene expression, gene diagnosis, genome sequencing.
The PCR technique derived:
(1) reverse transcription PCR (reverse transcription PCR, RT-PCR): RNA the reverse transcription PCR process combined with a PCR technique. I.e. under the action of reverse transcriptase, mRNA as a template for reverse transcription to generate cDNA, again using cDNA as template for PCR amplification.
RT-PCR primers are three kinds. a) random primers: unknown, long RNA having a hairpin structure or the reverse transcription, and the lowest specificity. b) Oligo (dT15-18): applicable to the polyA tail of the mRNA; c) specific primers: complementary to the sequence, an antisense oligonucleotide is applied to the case of the sequence is known.
Note: RNA ensuring no RNase-yl and contamination of genomic DNA.
Uses: Analysis of the transcription level of a gene construct efficient RNA transcription system.
(2) Quantitative RT-PCR (quantitative RT-PCR, QRT-PCR):
Established in the reaction system constant reference standard competitive RNA (RNA competitive reference standard, RNA-CRS), the mRNA was amplified with the target, the gene expression levels can be made semi-quantitative or quantitative analysis, which is RT -PCR.
(3) real-time quantitative PCR (real-time fluorescence quantitative PCR, FQ-PCR): a fluorescent labeling molecule in the PCR reaction, using the accumulated real-time monitoring of fluorescence signals throughout the PCR process, and finally quantifying the original template, the standard curve Methods.
Principle: fluoresces when double-stranded form.
Key Concepts:
Ct value: number of cycles required for the fluorescence threshold set hit the fluorescent signals during PCR amplification product is the threshold cycle Ct.
Dissolution profile: a double-stranded into single-stranded when the required temperature.
Amplification curve:
Gene inactivation techniques: the use of technology, DNA or mRNA level in a block or reduce the expression of virulence genes.
Commonly used gene inactivation techniques are: antisense nucleic acids, RNA interference (RNA interference, RNAi), ribozymes, triplex DNA.
RNAi: double-stranded RNA-induced post-transcriptional gene silencing.
Plasmid