Chapter 21 Techniques of Molecular Biology. Department of Food Science National Taiwan Ocean University

Transcription

1 Chapter 21 Techniques of Molecular Biology 吳彰哲 (Chang-Jer Wu) Department of Food Science National Taiwan Ocean University Introduction The methods of molecular biology depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves. Hybridization and sequencing understanding of the structure and base-pairing characteristics of DNA and RNA DNA cloning insight into the activities of DNA polymerase, restriction endonucleases and DNA ligase PCR isolate essentially any DNA segment 1

2 Electrophoresis through a gel separates DNA and RNA molecules according to size Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties. DNA gel mobility 1. DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole. 2. Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. 3. The mobility of circular DNA molecules is affected by their topological structures. 4. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled > linear > nicked or relaxed Gel matrix is an inserted, jello-like porous material that supports and allows macromolecules to move through. Polyacrylamide: has high resolving capability, and can resolve DNA/RNA that differ from each other as little as a single base pair/nucleotide. but can separate DNA over a narrow size range (up to a few hundred bp/nt). Agarose: a much less resolving power than polyacrylamide, but can separate DNA molecules of up to tens of kb DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EtBr). 2

3 Pulsed-field gel electrophoresis: 1. The electric field is applied in pulses that are oriented orthogonally to each other. 2. Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties. 3. Can effectively separate DNA molecules over kb and up to several Mb in length. Electrophoresis is also used to separate RNAs: RNA have a uniform negative charge as DNA does. RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. RNA can be treated with reagent such as glyoxal to prevent RNA base pairing, so that its mobility correlates with the molecular weight. Restriction endonucleases cleave DNA molecules at particular sites Why use endonucleases? To make large DNA molecules break into manageable fragments. Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences. RE used in molecular biology typically recognize short target sequences (4-8bp) that are usually palindromic, and cut at a defined sequence within those sequences. How to name a restriction endonuclease? EcoRI Escherichia coli R13 the 1 st such Species category strain enzyme found 3

4 How to estimate the frequency of the RE in a DNA molecule or genome? The random occurrence of the hexameric sequence: 1/4096 (1/4 6 ) The use of multiple REs allows different regions of a DNA molecule to be isolated. A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes. (1) Restriction enzymes differ in the recognition specificity: target sites are different. (2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ. (3) Restriction enzymes differ in the nature of the DNA ends they generate: blunt/flush ends, sticky/staggered ends. (4) Restriction enzymes differ in the cleavage activity. 4

5 DNA hybridization can be used to identify specific DNA molecules Hybridization: the process of base-pairing between complementary ssdna or RNA from two different sources. Probe: A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence. Labeling of DNA or RNA probes: Radioactive labeling: display and/or magnify the signals by radioactivity. Non-radioactive labeling: display and/or magnify the signals by antigen labeling - antibody binding - enzyme binding - substrate application (signal release). End labeling: put the labels at the ends 5 -end labeling using polynucleotide kinase (PNK) 3 -end labeling using terminal transferase Uniform labeling: put the labels internally Nick translation labeling of DNA Hexanucleotide primered labeling of DNA Hybridization probes can identify electrophoretically-separated DNAs and RNAs DNA (Southern blot) RNA (Northern blot) Genomic DNA preparation RNA preparation Restriction digestion - Denature with alkali - Agarose gel electrophoresis DNA blotting/transfer and fixation RNA Probe labeling Hybridization (temperature) Signal detection (X-ray film or antibody) Blot type Target Probe Applications Southern DNA DNA or RNA mapping genomic clones estimating gene numbers, etc Northern RNA DNA or RNA RNA sizes and abundance (gene expression level) 5

6 DNA cloning The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning. Processes of DNA cloning: Forming the recombinant DNA molecules by inserting your interested DNA fragments into a proper vector. (Require restriction enzymes and ligase) Transform the recombinant DNA molecules into competent cells. Propagation of the cells containing the recombinant DNA to form a clone, a set of identical cells containing the same recombinant DNA. Select the desired clones using the selective marker. 6

7 Three characteristics of vector DNAs : 1. They contain an origin of replication that allows them to replicate independently of the chromosome of the host. 2. They contain a selectable marker that allows cells that contain the vector to be readily identified. 3. They have single sites for one or more restriction enzymes (multiple cloning site), this allows DNA fragments to be inserted at a defined point within an otherwise intact vector. Plasmids: small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells. Contain an origin of replication, at least one selective marker and multiple cloning site. Example of selective marker: amp r gene encoding the enzyme β- lactamse which degrades penicillin antibiotics such as ampicillin. The commonly used plasmid are commonly small (~ 3 kb). Vector DNA can be introduced into host organisms by transformation Host organisms/cells: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Prokaryotic host: E. coli ( most cases) Transformation: 1. The process by which a host organism can take up DNA from its environment. 2. Some bacteria naturally have genetic competence (the ability to be transformed). 3. Some bacteria have genetic competence to do this, while E. coli must be treated with Ca 2+. 7

8 Libraries of DNA molecules can be created by cloning A DNA library is a population of identical vectors that each contains a different DNA insert. Genomic Library: the DNA inserts in a DNA library is derived from restriction digestion or physical shearing of the genomic DNA. cdna library: the cdna inserts in a DNA library is converted by reverse transcriptase and from the mrnas of a tissue, a cell type or an organism. cdna stands for the DNA copied from mrna. 8

9 Hybridization can be used to identify a specific clone in a DNA library Colony hybridization: the process by which a labeled DNA probe is used to screen a library. Transfer to nitrocellulose or nylon membrane Master plate Select positive from master plate Probe with 32 p-labled DNA complementary to gene of interest Expose to film Chemically synthesized oligonucleotides Short, custom-designed segments of DNA known as oligonucleotides. Protonated phosphoamidine is the pre-cursor which are chemically protected molecule used in nucleotide addition. Site-directed mutagenesis: Short DNA molecules up to 30 bases can be chemically synthesized efficiently and accurately. A custom-designed oligonucleotide can harbor a mismatch to a segment of cloned DNA can be used to create a directed mutation in cloned DNA. 9

10 The polymerase chain reaction (PCR) amplifies DNAs by repeated rounds of DNA replication in vitro The polymerase chain reaction (PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence. The PCR cycle: Denature: The target DNA (template) is separated into two stands by heating to 95 o C Annealing: The temperature is reduced to around 55 o C to allow the primers to anneal. Extension: The temperature is increased to 72 o C for optimal polymerization step which uses up dntps and required Mg 2+. Nested sets of DNA fragments reveal nucleotide sequences Two ways for sequencing: 1. DNA molecules (radioactively labeled at 5 termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. (Maxam and Gilbert chemical method, not widely used) 2. Chain-termination method (Sanger s method, widely used) 10

11 Chain-termination method: ddntps are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddntp is added to the 3 end. If one ddgtp is added to 100 dgtp, DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddgtp. When we spike DNA synthesis reactions with ddctp, ddatp, and ddttp, The lengths of DNA chains, terminated with the dideoxynucleotide indicated at the top of each lane, are determined by resolving on a polyacrylamide gel. Reading the gel from top to bottom gives the 5 to 3 sequence. 11

12 Sequenators are used for high throughput sequencing Automatic sequenator: Fluorescence chain-terminating nucleotides: fluorescence labeled ddntp Polymerase catalyzed Shotgun sequencing a bacterial genome 1. The genome was randomly sheared into many fragments with an average size of 1kb. 2. The pieces were cloned into plasmid recombinant DNA vector. 3. DNA was prepared from individual recombinant DNA colonies and separately sequenced on sequenators x sequence coverage means every nucleotide in the genome was sequenced ten times. 10 x coverage example: If the H. influenzae genome is 1.8 Mb, each read produces 600 bp of sequence, and 600 bp x 33,000 different colonies= 20 Mb. That is to say 33,000 colonies are picked to prepare plasmid for sequencing. 12

13 The shotgun sequencing permits a partial assembly of large genome sequences The key technical insights that facilitating the sequencing of the human genome was the reliance on a. automated shotgun sequencing (obtain sequence) b. then the subsequent use of computer to assemble the different sequences (analyze sequence, which is the rate-limiting step). Assembly Step 1: form contigs (A single contig is about 50,000 to 200,000 bp. ) Sophisticated computer programs have been developed that assemble the short sequences from random shotgun DNAs into larger contiguous sequences called contigs. Assembly Step 2: The paired-end strategy permits the assembly of larger scaffolds (1-2 Mb) The use of BACs (bacterial artificial chromosome) often permits the assignment of multiple contigs into a single scaffold by virtue of sharing several mesgabases. 13

14 The paired-end strategy permits the assembly of large-genome scaffolds A major limitation to producing large contigs is the occurrence of repetitive DNAs. One method that is used to overcome this difficulty is called paired-end sequencing. The specific cloning vector called a BAC (bacterial artificial chromosome) that can accommodate very large inserts, up to hundreds of kilobases of DNA. The $1000 human genome is within reach The 454 Life Sciences sequencing machine generates up to 400Mb of sequence information in a 4- hour run. 14

15 Protein purification The purification of individual proteins is critical to understanding their function. Although there are thousands of proteins in a single cell, each protein has unique properties, such as size, charge, shape, and in many instance, function, that make its purification somewhat different from others. Purification of a protein requires a specific assay to allow you to monitor your purification status, which include a measure of the function of the protein, use of the antibody of the protein. Incorporation assays are useful for monitoring the purification and function of many different enzymes catalyzing the synthesis of polymers like DNA, RNA, or proteins. The extraction of active proteins: Unlike DNA, which is very resilient to temperature, even moderate temperatures readily denature proteins once they are released from a cell. Most extract preparation and protein purification is performed at 4 o C. Cells can be lysed by detergent, shearing forces, treatment with low ionic salt or rapid changes in pressure. Protein can be separated from one another using column chromatography Column chromatography: Protein fractions are passed through glass columns filled with appropriately modified small acrylamide or agarose beads. There are various ways columns can be used to separate proteins on the basis of their charge or size, respectively. Ion exchange chromatography: The proteins are separated according to their surface charge. The beads are modified with either negative-charged or positive-charged chemical groups. Proteins bind more strongly requires more salt to be eluted. 15

16 Gel filtration chromatography: Proteins are separated by the basis of size and shape. The beads have a variety of different sized pores throughout. Small proteins can enter all the pores and can access more of column and take longer to elute. Large proteins can access less of the column and elute more rapidly. Affinity chromatography: If the target protein is known to establish a specific and highaffinity interaction with a specific protein/nucleic acids/small molecule, we can couple this specific partner of the target protein to the column and thus the target protein will be selectively bound to the column. This method is called affinity chromatography. Immunoaffinity chromatography: an antibody that is specific for the target protein is attached to beads. The primary difficulty is that frequently the antibody binds the target protein too tightly to be eluted with activity. Sometimes tags (epitopes) can be added to the N- or C- terminal of the target protein, using DNA cloning method, to make the fusion protein. This allows the modified fusion proteins to be purified using immunoaffinity purification and a heterologous antibody specific for the tag. Importantly, the binding affinity can change according to the condition. e.g. the concentration of the Ca 2+ in the solution. Immunoprecipitation: Attach the antibody to the bead, which is then used to precipitate a specific protein from a crude cell extract. It s a useful method to detect what proteins or other molecules are associated with the target protein. 16

17 Separation of proteins on polyacrylamide gels The native proteins have neither a uniform charge nor a uniform secondary structure. A protein is treated with the strong ionic detergent dodecyl sulphate (SDS) and a reducing agent, such as mercaptoethanol, the secondary, tertiary, and quarternary structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge. Sometimes, mercaptoethanol is need to break the disulphide bond. Electrophoresis in the presence of SDS can resolve mixtures of proteins according to the length of individual polypeptide chains. After electrophoresis, the proteins can be visualized with a stain, such as Coomassie brilliant blue. Antibodies are used to visualize electrophoretically separted proteins Immunoblotting (Western blotting): Proteins from the PAGE gel can be transferred to a membrane, followed by a western analysis of the target protein by a corresponding antibody. 17

18 Protein molecules can be directly sequenced Methods for determining protein sequence: Edman degradation and tandem mass spectrometry. Due to the vast resource of complete or nearly complete genome, the determination of even a small stretch of protein sequence is sufficient to identify the gene. Edman degradation: A chemical reaction in which the amino acid s residues are sequentially released for the N-terminus of a polypeptide chain, and specifically modified by a chemical reagent called phenylisothiocyanate (PITC). The identity of the released amino acid derivative can be easily determined by its elution profile using a column chromatography method called High Performance Liquid Chromatography (HPLC). The whole process can be carried out in an automatic protein sequencer. Step 1: modify the N-terminal amino with PITC, which can only react with the free α-amino group. Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact. Step 3: identify the released amino acids by HPLC. 18

19 Tandem mass spectrometry (MS/MS): Determine regions of protein sequence. A method in which the mass of very small samples of a material can be determined with great accuracy. Material travels through the instrument in a manner that is sensitive to its mass/charge ratio. Step 1: digest your target protein into short peptides. Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate. Step 3: capture the individual peptide and fragmented into all the component peptide. Step 4: determine the mass of each component peptide. Step 5: deconvolution of these data and the sequence will be revealed. Proteomics Proteomics is concerned with the identification of the full set of proteins produced by a cell or a tissue under a particular by a particular set of conditions. Three principal methods: Two-dimensional gel electrophoresis for protein separation. Mass spectrometry for the precise determination of molecular weight and identify of a protein. Bioinformatics for assigning proteins and peptides to the predicted products of proteincoding sequence in the genome. 19

20 Protein-protein interactions can yield information about protein function Proteomics is also concerned with identifying all the proteins that associate with another protein in a cell to generate what are called interactomes. One method for determining proteinprotein interactions is the yeast twohybrid assay, whereby the protein of interest serves as bait and a library of proteins can be test as potential prey. 20

21 The electrophoretic mobility of DNA is altered by protein binding Electrophoretic mobility-shift assay (EMSA): a short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. Then, samples of labeled nucleic acid, with and without being incubated with the extract, are run on a gel. The DNAprotein complexes are shown by the presence of slowly migrating bands. EMSA can be used to detect protein- DNA interaction. DNA-bound protein protects the DNA from nucleases and chemical modification Protection footprint: identify the actual region of DNA sequence with which the protein interacts. Nuclease protection footprint:1. The protein protects DNA from attack by nuclease. 2. Treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule. Chemical protection footprint: The protein protect bases in the binding site from basespecific chemical reagents that give rise to backbone cuts. 21

22 Chromatin immunoprecipitation can detect protein association with DNA in the cell Chromatin immunoprecipitation (ChIP): is a powerful technique to monitor protein-nucleic acid interactions in the cell. 1. Formaldehyde is added to living cells, cross-linking DNA to proteins, protein to protein. 2. The cross-linked cells are lysed and the DNA is broken into bp fragments. 3. Using an specific antibody for transcription regulator, the fragment of DNA attached to that protein can be separated by immunoprecipitation (IP). 4. The cross-linking between protein and DNA reversed, allowing analysis of the DNA sequences that are present in the IP. In vitro selection can be used to identify a protein s DNA- or RNA-binding site In vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) involves the use of the sequence specificity of the protein to probe a diverse library of oligonucleotides. By characterizing the enriched DNA, the sequences that bind tightly to the protein can be identified. 22