Molecular Techniques PDF

Introduction to Some Molecular Biology Techniques Methods for assessing : Individual gene structure and expression Presented by Dr Ghada KHAWAJA  PROBLEM : Metamorphosis in amphibian is a process involving many radical molecular, morphological and biochemical changes tra...

Introduction to Some Molecular Biology Techniques Methods for assessing : Individual gene structure and expression Presented by Dr Ghada KHAWAJA  PROBLEM : Metamorphosis in amphibian is a process involving many radical molecular, morphological and biochemical changes transforming an aquatic larvae to a terrestrial animal. This phenomenon is triggered by thyroid hormones. Thyroid hormones act through thyroid hormone receptors (THR) which are encoded by α and β THR genes. Determining the role of THR genes can aid to better understanding the phenomenon. Let’s begin with THR α gene of Xenopus laevis, an African frog. Some questions have to be answered :  What is the structure of THR α gene ?  How is it expressed (at which time, in which tissues, at what amount) ?  How does it interact with other genes to control metamorphosis ?  SOME METHODS ARE INTRODUCED BELOW AS SUGGESTED SOLUTIONS. OTHER STRATEGIES AND METHODS COULD BE IMAGINED  Firstly, the genetic material of X. laevis is extracted from frog tissue  DNA/RNA extraction methods  Purified DNA/RNA are qualitatively and quantitatively analyzed before further investigation  Electrophoresis and Spectrophotometer-based methods What is the structure of Xenopus laevis THR α gene?  THR α gene is isolated and amplified from genomic DNA/mRNA  Cloning, PCR (Polymerase Chain Reaction), RT-PCR methods  The gene is detected by Southern blotting  Finally, the nucleotide sequence of THR α gene is determined by Sequencing methods How is it expressed (at  Differential expression of THR α gene is evaluated by Northern which time, in which tissues, at what amount) ? blotting, or Real-time RT-PCR What are the other genes implied in X. laevis  Identification of other genes involved  DNA Microarray metamorphosis ? How are Analysis they expressed? NUCLEIC ACID EXTRACTION METHODS According to the nature of sample (animal, plant tissues or microbial organisms, …), and the kind of nucleic acids to be extracted, different extraction agents are used. Nevertheless, DNA/RNA extraction methods are usually composed of three steps which could be separated or combined. Step 1 : Cell membrane lysis Tissues are mechanically (ground) and/or enzymatically broken. Cell membrane are lyzed by detergents (SDS,..) or chaotropic agents, such as Guanidinium thiocyanate which is commonly used for RNA extraction. Step 2 : Protein elimination A Phenol:Chloroform:Isoamyl alcohol solution is usually used to denature proteins which are consecutively eliminated by centrifugation. Some resins could also be used to eliminate proteins. This step could be combined with step 1 to simultaneously lyzing membranes and eliminating proteins. Acidic Phenol is used for RNA extraction instead of basic Phenol used in DNA extraction. Step 3 : Obtention of purified nucleic acids. Many approaches can be used: DNA/RNA precipitation by Ethanol/Isopropanol combined with salt. DNA/RNA are recovered by centrifugation and redissolved. Adsorption of DNA/RNA on a matrix (silica,…) followed by washing and DNA/RNA elution. The purified nucleic acids must then be analyzed to assess their amount as well as their quality (integrity, purity). The two common methods used include Electrophoresis and Spectrophotometric measure of Optical Density (OD). Electrophoresis methods are usually considered as qualitative analysis whereas Spectrophotometric measures are mostly used as quantitative analysis. Nevertheless, combined with other methods they can be used indifferently for qualitative as well as for quantitative analysis. ELECTROPHORESIS Nucleic acids which are negatively charged in solution will migrate in an electric field toward the positive pole. The electric field is usually established in a semi-solid matrix (gel) made of high-molecular weight polysaccharides such as agarose or acrylamide. Electrophoresis are used to separate different nucleic acid fragments in a mixture. During electrophoresis, nucleic acids have to “creep” through the network established by agarose or acrylamide molecules. The migration rate of nucleic acids depends on many factors : their size, the gel concentration which determines “loose” or “tighten” meshes, the voltage,… Agarose gels are commonly used for separation and detection of nucleic acid fragments. Acrylamide (usually in the form of polyacrylamide) gels which have high resolution capacity are used for specific purposes, e.g. Sequencing gels where a one-nucleotide difference in size can be recognized. Nucleic acids are detected after electrophoresis by staining with some fluorescent agents (Ethidium bromide, SYBrgreen,..). These agents are incorporated in nucleic acids and fluoresce under UV light. Molecular weight markers are used in electrophoresis for detecting the size of the desired fragment. Electrophoretic analysis can answer the question about the presence and the integrity (molecular size) of nucleic acid fragments. It also contributes to identify a particular DNA/RNA fragment when combined with other methods such as Molecular hybridization. SPECTROPHOTOMETRIC ANALYSIS Light particles entering a solution will be absorbed by molecules present in the solution. Light particles absorption increases with increasing numbers of molecules present in the solution. Optical density (OD) measure is performed by a spectrophometer, based on the difference of light intensity before and after entering a solution containing the molecules to be quantitated. The intensity decrease after light entering a solution is due to absorption by these molecules. In nucleic acids it is purines and pyrimidines which absorb light ; absorption is maximal at a wavelength of 260 nm. Nucleic acids concentration can be calculated based on their OD values as follows : 1 OD260 nm ≈ 50 µg/ml double-stranded nucleic acids ≈ 40 µg/ml single-stranded nucleic acids ≈ 20 µg/ml oligonucleotides Furthermore, the purity (protein contamination-free) of nucleic acid solutions can be assessed by the ratio of OD260 nm/ OD280 nm values. A ratio of 1.8 – 2 shows good DNA purity whereas an inferior value means protein contamination. OD measurement of nucleic acid solutions are used to calculate their concentration as well as their purity before further studies. The purified nucleic acids are now ready for further use. They can be cut and ligated, or replicated. All these activities are performed by enzymes – nucleases, ligases, polymerases. UTILIZATION OF SOME ENZYMES IN MOLECULAR TECHNIQUES  To isolate a particular gene, two methods are commonly used : PCR (Polymerase Chain reaction) and Molecular Cloning. Both methods are based on the amplification of gene copies. In Molecular cloning, also called in vivo amplification, gene copies are replicated by living cells. Molecular cloning must be followed by other methods (Molecular hybridization or PCR) if a specific gene has to be isolated. PCR, an in vitro amplification of gene copy number, is performed in lab tubes. The amplification is gene-specific thus PCR products consist of large amount of the gene of interest. PCR (POLYMERASE CHAIN REACTION) PCR consists of repeated rounds of DNA replication resulting in important amplification of a specific DNA fragment. PCR is composed of many cycles, each includes three steps : (1) Denaturation, the two DNA strand are separate by heating, (2) Annealing, target-specific primers (forward and reverse) hybridize to complementary sequence in the DNA target, (3) Elongation, thermophilic DNA polymerases synthesize complementary strands from annealed primers. According to theoretical calculations, amplification can result in 106 copies from the initial one after 30 cycles of PCR. PCR can be used to obtained a large amount of a particular DNA fragment for further manipulations (cloning, sequencing), or to detect a specific target DNA at very low concentration in samples. Many PCR-based methods were developed : RT-PCR combining reverse transcription with PCR to amplifying RNA, quantitative PCR (qPCR or real-time PCR) allowing the quantitation of initial DNA target number, in situ PCR amplifying target DNA in tissues without prior extraction, RAPD using random primers to amplifying large regions of genomic DNA, … qPCR (REAL-TIME PCR) In this technique, fluorescent particles are integrated into the newly synthesized double-stranded DNA during the amplification process. The increasing amount of fluorescent signals reflect the increasing amount of replicated DNA and is recorded at each moment of the amplification reaction. Real-time PCR is used to quantitate DNA content in a sample or to compare DNA amount in different samples. RNA can be quantitated by real-time RT-PCR combining a reverse transcription with the real-time PCR. Fluorescent agents are of two types : (1) DNA binding dyes, e.g SYBrGreen, which fluoresce when intercalating into doubled-strand DNAs, (2) Fluorescent agents used to label target-specific probes, e.g Hydrolysis probes, Hybridization probes, Molecular beacon, … Example of target-specific probes, Hydrolysis or Taqman Probe. Taqman probe is labelled at the 5’end by a reporter fluorophore and at the 3’end by a quencher fluorophore which “quenches” the reporter. During elongation step, Taq polymerase displaces the annealed probe, releasing the reporter fluorophore which is no longer quenched by the quencher. Each signal emitted by the released reporter group is equivalent to a newly synthesized strand and can be recorded precisely at any moment of the amplification process, where the term “real-time”. How can the initial amount of DNA in the sample be calculated ?  qPCR (REAL-TIME PCR) (continued) An important notion of real-time PCR is the Ct (Cycle threshold). Ct is the amplification cycle at which the signal is recognized as specific signal, arising from the non specific background context. To calculate the initial amount of DNA in the sample, a standard curve is established with predetermined concentration of a standard DNA. The initial concentration of target DNAs in samples can be determined from the established standard curve  MOLECULAR CLONING Molecular cloning refer to the amplification of DNA fragments by cellular DNA replication system. The approach consists of introducing the DNA fragment into a host cell and inducing its replication. To create a self-replicating DNA molecule, the target DNA (insert) is incorporated into a vector ; the vector become a recombinant vector. Vectors are DNA molecules having some characteristics :  They contain an origine of replication which allows them to replicate independently in host cell Example of a vector :  Ori : origin of replication  They contain many restriction sites which are substrate for  Amp R : Ampicillin- restriction enzymes. resistant gene  MCS : Multicloning site (PCS  They contain markers allowing identification of cells being -polycloning site, transformed with these vectors, such as antibiotic resistance genes polylinker), sequence which permit transformed cells to grow in media containing containing many restriction antibiotics. sites  Vectors can bear other sequences with specific utility ; e.g vectors  Lac promoter/lac Z : region containing promoters are used to express cloned genes into RNAs. flanking the MCS, allowing the detection of cells Depending on research purposes, vectors of different kinds can be containing the recombinant used : plasmid, cosmid, phage, animal virus, plant virus, BAC (Bacterial vector Artificial Chromosome), YAC (Yeast Artificial Chromosome), MAC  T7/SP6 promoter : allowing (Mammalian Artificial Chromosome), … the expression of cloned gene into RNAs  MOLECULAR CLONING (continued) Selection of a specific clone is made by a molecular hybridization process performed in colonies.  MOLECULAR HYBRIDIZATION The pase-pairing of two single strand DNAs from different source but having complementary sequence is called Molecular Hybridization. In Hybridization, a probe is used to detect a specific nucleic acid sequence. Probes have some charateristics : (1) Having sequence complementarity with the target sequence, (2) Being chemically or radioisotopically labelled. Probes could be single-stranded DNA, RNA or oligonucleotide. Based on the location of target sequences, hybridization can be classified as :  Hybridization in liquid phase between targets and probes present in solution  Hybridization on solid substrate : the target sequences are fixed on a subtrate such as membrane, plate, slides, … , e.g Southern/Northern/Dot blot hybridization, technique of microarray, …  In situ hybridization : taking place directly on tissues without prior extraction of the target sequences. Hybridization in colonies can be considered as an in situ hybridization process. Bacterial cell membrane are lyzed and the recombinant vectors present inside the cells are hybridized with a probe. Southern and Northern blot hybridization are introduced as follows SOUTHERN/NORTHERN BLOTTING SOUTHERN/NORTHERN BLOTTING (continued)  WESTERN BLOTTING Western blotting entails separation of proteins by size on a sodium dodecyl sulfate (SDS) gel, transfer to a nitrocellulose membrane, and detection of proteins of interest using antibodies.  DIDEOXY SEQUENCING (METHOD OF SANGER ) The principle of Dideoxynucleotide sequencing (or method of Sanger) is based on random incorporations of dideoxynucleotides into the ongoing replicated DNA strand. Lacking the C3’-OH, a dideoxynucleotide (ddNTP), e.g dideoxyadenine – ddATP, or ddCTP, ddGTP, ddTTP, can not participate in the formation of phosphodiester bonds. Thus, at any time a ddNTP is randomly incorporated in the elongated strand, DNA synthesis is immediately interrupted. To determine the nucleotide sequence of a DNA fragment, 4 reactions are established, each including all the components necessary to DNA replication plus 1 of the 4 ddNTPs. At the end of replication process, the four reactions are analyzed by polyacrylamide gel electrophoresis. A portion of radiolabeled dATP is added to each replication reaction so that the DNA sequence can be read after an autoradiography of the polyacrylamide gel DIDEOXY SEQUENCING (continued)  DIDEOXY SEQUENCING (continued) Automatic sequencing was developed based on the method of Sanger. Automatic sequencing is performed in a sequencer and uses, instead of radiolabelling, different fluorescent dye-labelled ddNTPs. The terminating replication reaction are then analyzed by electrophoresis. During electrophoresis, migrating DNA fragments are induced and emit different fluorescent signals when crossing a detector. The signals are recorded and reflect the terminal ddNTP of each DNA fragments. The overall result is the nucleotide sequence to be determined (below). Improvements of automatic sequencing involve more efficient sequencing enzymes, and particularly more efficient electrophoretic analysis, e.g capillary electrophoresis. The great beneficiary of automatic sequencing are Genome projects, the most important of which is the Human Genome Project.  Metamorphosis is a highly complicated process involving numerous genes interacting with each other. “What are the other genes, besides THR α gene, also included in the process of metamorphosis in X. laevis ?” By means of the previously described methods, genes are analyzed individually, one after another. Analysis are slow and do not provide an overview of the whole biological network.  DNA Microarray Genome-wide transcription analysis is performed using labeled cDNA from experimental samples hybridized to a microarray containing sequences from all ORFs of the organism being used. DNA microarrays comprise known DNA sequences spotted or synthesized on a small chip. Gene expression arrays are used to detect the levels of all the expressed genes in an experimental sample. DNA Microarray (continued) SUMMARY Individual gene structure and expression can be determined by sequential or simultaneous utilization of the following methods :  Nucleic acid extraction methods  Qualitative and quantitative analysis based on Electrophoresis and Spectrophotometric measures  Molecular Cloning  PCR and other derivative techniques  Molecular Hybridization – Southern/Northern Blot, Dot Blot, in situ Hybridization, Western Blot  DNA Sequencing  …..  Functional Genomics using methods such as DNA microarray