【共享】简明扼要的一些重要分子生物学概念（in English）！
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Nucleic Acids
Gel electrophoresis
Agarose Gel Electrophoresis
Pulse-field Gel Electrophoresis
Polyacrylamide Gel Electrophoresis
Restriction Enzyme (RE) Digestion
What are RE?
Enzymes that cut dsDNA, usually palindromic sequences. RE recognize 4-8bp.
A RE that recognizes 6bp cleaves on average every 4kb.
Blunt cutters and cutters leaving overhangs (3í or 5í)
Southern Blot (named after Edward Southern)
Probe preparation
Introduce labeled precursor into newly synthesized DNA strand
Label the end of the DNA strand with polynucleotide kinase and g32P dATP.
DNA to be probed
Run RE digested DNA on agarose gel
Denature DNA in gel
Transfer DNA on to a positively charged nitrocellulose membrane (for example)
Probe the DNA on the blot with the labeled probe.
Note: Northern Blot is similar to the Southern blot, but the nucleic acid analyzed is the RNA molecule.
DNA Cloning
ìThe ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.î
A vector is usually a plasmid vector or phage vector that provides the information necessary to propagate the cloned DNA in the cell (bacterial cell or yeast cell, for example). Vectors have an origin of replication, a selectable marker and ideally, multiple cloning sites. Some vectors are expression vectors. These expression vectors have transcriptional promotores immediately adjacent to the site of insertion.
An insert is the DNA of interest.
Once a construct (vector + insert) is made, propagation of the construct can be made by introducing the construct into a bacterial host, a process called transformation
Bacteria need to have genetic competence before they can be transformed. E. coli, for example, is rendered competent by treating with calcium ions. The inefficiency of transformation also ensures that, in most cases, each cell receives only a single molecule of DNA.
Libraries of DNA
A DNA library is a population of identical vectors that each contains a different DNA insert.
The simplest libraries are derived from total genomic DNA cleaved with a RE. These are called genomic libraries. This type of library is useful when generating DNA for sequencing a genome.
To look for coding sequences, a cDNA library is constructed. To make this library, mRNA from the organism is reverse transcribed into cDNA (copy DNA). These cDNA are then ligated into the vector.
To screen a library with a labeled probe, colony hybridization is performed.
Polymerase Chain Reaction (PCR)
Denaturation of the dsDNA template.
Annealing of synthetically made oligonucleotide primers.
Extension by thermally stable DNA Polymerase using dNTPs.
DNA sequencing - Sanger dideoxynucleotide triphosphate
The ddNTP are added at a ratio of 1 ddNTP to 100 dNTPs.
Labeled primer needed in the 5íto 3í orientation and a template in the 3í to 5í orientation.
DNA Polymerase needed.
Shotgun Sequencing
Genomic DNA is sheared into many fragments of about 1 kb. These pieces of genomic DNA are cloned into a plasmid. DNA is prepared from individual recombinant DNA colonies and separately sequenced on Sequenators by the dideoxy method. In principle, every nucleotide in the genome is sequenced ten times. The sequencial assemvly of such short DNA sequences ultimately leads to a single continuous assembly, also called a contig.
Shotgun Sequencing of large genome sequences
The average human chromosome is 150Mb.The 600 bp of Dna sequence provided by a typical sequnceing reaction represent only 0.0004% of a typical chromosome.
Human Genome sequencing
DNA was prepared from each of the 23 chromosomes and reduced into pools of libraries of small fragmaens using small gauge pressurized needles. Fragments of 1 and 5 Kb were cloned into ìnormalî bacterial plasmids. The 100 kb fragments were cloned into bacterial artificial chromosome (BAC) .
A cluster of 100 384-column automated sequenceing machines cqan genrate tenfold coverage of human chromosome in just 3 weeks.
Individual contigs are typically composed of 50 to 200 Kb. This is still far short of a tyical human chromosome. However, such contigs are useful for analyzing compact genoms. The juamn genome contains an average of one gene every 100Kb, so a typical contig is oftern insufficient to capture an entire gene,, let alone a series of linked genes. Short contigs are assembled into larger scaffolds that are typically 1-2 Mb in length.
To find protein coding genes, open-reading frames are sought. This is an easy feat when looking at bacterial and simple eukaryotic genomes, however, bioinformatics is needed to determin protein coding sequences in higher eukaryotes because of repeated sequences large introns, etc.
Microarray
Please look at the following link:
http://www.gene-chips.com/
Proteins
Proteins are obtained from cell extracts by a series of fractionation steps via centrifugation.
Separation by Column Chromatography
Size exclusion chromatography-separates proteins by size.
Ion exchange chromatography- separates proteins by charge.
Affinity chromoatography-separates proteins according to the affinity the protein has for a particular functional group.
Immunoprecipitation- bead linked-antibodies specific for a protein precipitate the specified protein out of a mixture of proteins.
Gel Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
2D-Gel Electophoresis- first dimension is isoelectrofocusing (separates proteins according to their isoelectric point (pI)). Second dimension uses SDS-PAGE to separate proteins according to their size. Thus, by 2D-Gel electrophoresis proteins are separated according to their pI and MW.
Protein detection in gel: To detect proteins on the gel, commassie blue stain can be used. Alternatively, radioactivity or fluorescence can be used.
Western Blot and Immunoblot. Western blot is the process of transferring protein from a PAGE or SDS-PAGE to a membrane to immobilize the proteins for immunoblotting (antibody detection of a specifc protein) Proteomics is concerned with the identification of the full set of proteins produced by a cell or tissue under a particular set of conditions.
Protein sequencing- Edman degradation. This technique relies of de4ivatizing the N terminal amino acid with phenylisothiocyanate (PITC). This derivatized amino acid is then cleaved off the polypeptide by treatment with acid under conditions that do not destroy the remaining proteins. The identity of the released amino acid derivative can be easily determined by its elution profile on HPLC. Each amino acid has a specific retention time. By sequencing about 15 amino acids, the identity of a protein can be determined.
Tandem mass spectrometry (MS/MS) is also used to determine protein sequence. The protein of interest is usually digested into short peptides by digestion with a specific protease. This mixtutre of peptides is subjected to mass spectrometry and each individual peptide witll be separated from the others in the mixture by its mass/charge ratio. The individual peptides are then captured and fragmented into all the c9ponent peptides, and the amss of each of these component fragments is then determined.
Proteomics
Proteomics is concerned with the identification of the full set of proteins produced by a cell or tissue under a particular set of conditions
Proteomics is based on 3 methods: 2D gels, mass spec and bioinformatics.
While each of our cells have the same genome, each cell type has a different proteome. In addition to cell to cell variation, any given cell will change its proteome over time. As you grow older, your proteomes change. When you have an infection, they are altered to meet the new challenge. Medications, exercise, diet- your proteomes change in response to fluctuations in the intracellular and extracellular environments.
Gel electrophoresis
Agarose Gel Electrophoresis
Pulse-field Gel Electrophoresis
Polyacrylamide Gel Electrophoresis
Restriction Enzyme (RE) Digestion
What are RE?
Enzymes that cut dsDNA, usually palindromic sequences. RE recognize 4-8bp.
A RE that recognizes 6bp cleaves on average every 4kb.
Blunt cutters and cutters leaving overhangs (3í or 5í)
Southern Blot (named after Edward Southern)
Probe preparation
Introduce labeled precursor into newly synthesized DNA strand
Label the end of the DNA strand with polynucleotide kinase and g32P dATP.
DNA to be probed
Run RE digested DNA on agarose gel
Denature DNA in gel
Transfer DNA on to a positively charged nitrocellulose membrane (for example)
Probe the DNA on the blot with the labeled probe.
Note: Northern Blot is similar to the Southern blot, but the nucleic acid analyzed is the RNA molecule.
DNA Cloning
ìThe ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.î
A vector is usually a plasmid vector or phage vector that provides the information necessary to propagate the cloned DNA in the cell (bacterial cell or yeast cell, for example). Vectors have an origin of replication, a selectable marker and ideally, multiple cloning sites. Some vectors are expression vectors. These expression vectors have transcriptional promotores immediately adjacent to the site of insertion.
An insert is the DNA of interest.
Once a construct (vector + insert) is made, propagation of the construct can be made by introducing the construct into a bacterial host, a process called transformation
Bacteria need to have genetic competence before they can be transformed. E. coli, for example, is rendered competent by treating with calcium ions. The inefficiency of transformation also ensures that, in most cases, each cell receives only a single molecule of DNA.
Libraries of DNA
A DNA library is a population of identical vectors that each contains a different DNA insert.
The simplest libraries are derived from total genomic DNA cleaved with a RE. These are called genomic libraries. This type of library is useful when generating DNA for sequencing a genome.
To look for coding sequences, a cDNA library is constructed. To make this library, mRNA from the organism is reverse transcribed into cDNA (copy DNA). These cDNA are then ligated into the vector.
To screen a library with a labeled probe, colony hybridization is performed.
Polymerase Chain Reaction (PCR)
Denaturation of the dsDNA template.
Annealing of synthetically made oligonucleotide primers.
Extension by thermally stable DNA Polymerase using dNTPs.
DNA sequencing - Sanger dideoxynucleotide triphosphate
The ddNTP are added at a ratio of 1 ddNTP to 100 dNTPs.
Labeled primer needed in the 5íto 3í orientation and a template in the 3í to 5í orientation.
DNA Polymerase needed.
Shotgun Sequencing
Genomic DNA is sheared into many fragments of about 1 kb. These pieces of genomic DNA are cloned into a plasmid. DNA is prepared from individual recombinant DNA colonies and separately sequenced on Sequenators by the dideoxy method. In principle, every nucleotide in the genome is sequenced ten times. The sequencial assemvly of such short DNA sequences ultimately leads to a single continuous assembly, also called a contig.
Shotgun Sequencing of large genome sequences
The average human chromosome is 150Mb.The 600 bp of Dna sequence provided by a typical sequnceing reaction represent only 0.0004% of a typical chromosome.
Human Genome sequencing
DNA was prepared from each of the 23 chromosomes and reduced into pools of libraries of small fragmaens using small gauge pressurized needles. Fragments of 1 and 5 Kb were cloned into ìnormalî bacterial plasmids. The 100 kb fragments were cloned into bacterial artificial chromosome (BAC) .
A cluster of 100 384-column automated sequenceing machines cqan genrate tenfold coverage of human chromosome in just 3 weeks.
Individual contigs are typically composed of 50 to 200 Kb. This is still far short of a tyical human chromosome. However, such contigs are useful for analyzing compact genoms. The juamn genome contains an average of one gene every 100Kb, so a typical contig is oftern insufficient to capture an entire gene,, let alone a series of linked genes. Short contigs are assembled into larger scaffolds that are typically 1-2 Mb in length.
To find protein coding genes, open-reading frames are sought. This is an easy feat when looking at bacterial and simple eukaryotic genomes, however, bioinformatics is needed to determin protein coding sequences in higher eukaryotes because of repeated sequences large introns, etc.
Microarray
Please look at the following link:
http://www.gene-chips.com/
Proteins
Proteins are obtained from cell extracts by a series of fractionation steps via centrifugation.
Separation by Column Chromatography
Size exclusion chromatography-separates proteins by size.
Ion exchange chromatography- separates proteins by charge.
Affinity chromoatography-separates proteins according to the affinity the protein has for a particular functional group.
Immunoprecipitation- bead linked-antibodies specific for a protein precipitate the specified protein out of a mixture of proteins.
Gel Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
2D-Gel Electophoresis- first dimension is isoelectrofocusing (separates proteins according to their isoelectric point (pI)). Second dimension uses SDS-PAGE to separate proteins according to their size. Thus, by 2D-Gel electrophoresis proteins are separated according to their pI and MW.
Protein detection in gel: To detect proteins on the gel, commassie blue stain can be used. Alternatively, radioactivity or fluorescence can be used.
Western Blot and Immunoblot. Western blot is the process of transferring protein from a PAGE or SDS-PAGE to a membrane to immobilize the proteins for immunoblotting (antibody detection of a specifc protein) Proteomics is concerned with the identification of the full set of proteins produced by a cell or tissue under a particular set of conditions.
Protein sequencing- Edman degradation. This technique relies of de4ivatizing the N terminal amino acid with phenylisothiocyanate (PITC). This derivatized amino acid is then cleaved off the polypeptide by treatment with acid under conditions that do not destroy the remaining proteins. The identity of the released amino acid derivative can be easily determined by its elution profile on HPLC. Each amino acid has a specific retention time. By sequencing about 15 amino acids, the identity of a protein can be determined.
Tandem mass spectrometry (MS/MS) is also used to determine protein sequence. The protein of interest is usually digested into short peptides by digestion with a specific protease. This mixtutre of peptides is subjected to mass spectrometry and each individual peptide witll be separated from the others in the mixture by its mass/charge ratio. The individual peptides are then captured and fragmented into all the c9ponent peptides, and the amss of each of these component fragments is then determined.
Proteomics
Proteomics is concerned with the identification of the full set of proteins produced by a cell or tissue under a particular set of conditions
Proteomics is based on 3 methods: 2D gels, mass spec and bioinformatics.
While each of our cells have the same genome, each cell type has a different proteome. In addition to cell to cell variation, any given cell will change its proteome over time. As you grow older, your proteomes change. When you have an infection, they are altered to meet the new challenge. Medications, exercise, diet- your proteomes change in response to fluctuations in the intracellular and extracellular environments.
