Research methodology Chapter 6 summary
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Molecular biology techniques allow researchers to analyze and modify DNA and RNA. Key developments included the discovery of restriction endonucleases and DNA ligase in the 1960s, enabling the cutting and joining of DNA fragments. This founded recombinant DNA technology and led to the development of vectors like plasmids and viruses for inserting exogenous DNA into host cells. Techniques like PCR, DNA sequencing, microarrays, and transgenic organisms now allow comprehensive analysis of genomes and gene expression, advancing fields like genetics and medicine.
Research methodology Chapter 6 summary
- 1. RESEARCH METHODOLOGY CHAPTER 6: METHODS OF MOLECULAR BIOLOGY
- 2. 6.1 INTRODUCTION Molecular biology can be defined as the scientific discipline covering the level between chemistry and biochemistry on the one side and cell biology, histology, anatomy and physiology on the other, intertwined with and overlapping the levels on each side.
- 3. RECOMBINANT TECHNOLOGY The foundation was laid in the 1960s, with the discoveries of: • Restriction endonuclease (RE) enzymes - to cut DNA at specific sites into shorter fragments amenable to analysis • The techniques of DNA denaturation/renaturation and hybridization - by which specific pieces of DNA could be identified and pulled out from a large pool of cut fragments • The enzyme DNA ligase - allowed joining of fragments together into new combinations.
- 4. 1970’S DNA cloning was made feasible in the early 1970s. Marked the birth of recombinant DNA technology.
- 5. 6.2 RESTRICTION ENDONUCLEASES Endonucleases are enzymes that cleave the phosphodiester bonds of the DNA or RNA backbone Some restriction endonucleases cut the two strands immediately opposite one another, generating blunt ends, staggered cuts, generating single-stranded overhangs referred to as sticky ends
- 6. VECTORS* Plasmid vectors Vector is a 1. Carrier (medicine) 2. DNA molecules with the capacity to replicate when introduced into host cells and onto which we can load pieces of DNA to be amplified (Molecular Biology) Polylinker or multiple cloning site are several different recognition sites put one after the other within a small region. A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species Three elements are shared by all plasmid vectors 1. An origin of replication, i.e. a sequence necessary for replication in the bacterial host; 2. A cloning site containing recognition sequences for one or more restriction enzymes, wherein the desired DNA fragment can be inserted. 3. A selection marker - a gene conferring a phenotype that can be selected for or against, usually a sequence conferring resistance to antibiotics
- 7. Viral Vectors Viruses are protein-coated infectious DNA or RNA particles with naturally evolved mechanisms for unassisted entry into host cells. Bacteriophages are viruses that infect and replicate in bacteria Specialized to host cells they can infect ( tropism) Viral vectors : Advantage - to transfer genes into intact multicellular organisms. E.g. rats Disadvantage - random sites of genomic integration, they generate the hazard of unwanted spread. Transferring DNA into host cells – Conjugation - process for genetic exchange between bacteria Transduction – Transfer genetic information between cells Transformation is the genetic alteration of bacterial cells as a result of uptake of foreign DNA or RNA. Transfection - Non-viral transfer of DNA into eukaryotic cells Transformation - refers to the changes occurring when eukaryotic cells become malignant Bacteria can spontaneously take up non-viral nucleic acids from the surroundings. Methods used to regulate uptake are; Electroporation, or by adding Ca2 or other divalent cations to the medium
- 8. To insert non-viral DNA into eukaryotic cells the used techniques are: 1. Electroporation, 2. Injection directly into the cell (microinjection) 3. Complexing the DNA with molecules that facilitate the DNA transfer In bacterial transformation, the new DNA is normally not integrated into the bacterial chromosome, but remains as an extrachromosomal plasmid In eukaryotic cells, transfection may be transient or stable. Transient transfection, many copies of the DNA are transferred into each cell, where they remain extrachromosomally and are expressed only for a short period, usually a few days Stable transfection, the foreign DNA, which is either integrated into chromosomal DNA or stays extrachromosomally, is maintained by selection for resistance to cytotoxic drugs
- 9. Escherichia coli The laboratory workhorse Its full genome sequence (approx. 4.6 million bp) was reported in 1997 Divides rapidly Exclusively the bacterial species used in DNA technology strains divide every 20–30 minutes then lag phase. ( Continuous division is log phase)
- 10. 6.3 THE TECHNIQUES OF DNA DENATURATION/RENATURATION AND HYBRIDIZATION DNA AND RNA: ISOLATION, IDENTIFICATION, SYNTHESIS AND ANALYSIS Isolation of DNA and RNA DNA and RNA are negatively charged Not soluble in organic solvents (phenol and chloroform) Soluble in water The phenol/chloroform isolation method disadvantages: It is time consuming involves flammable and toxic liquids Replaced by chromatographic methods Chromatographic method steps ( DNA/RNA Purification) 1. Anion-exchange resins consist of positively charged solid particles, to which the highly negatively charged DNA and RNA molecules binds strongly. 2. Sugars and proteins, which bind with moderate affinity, can be washed off the resin 3. Highly purified nucleic acids are then eluted using buffers with the appropriate pH and salt concentration
- 11. Purification Method ( Density Difference) DNA/RNA purification is based on differences in densities between RNA, supercoiled DNA, linear DNA, proteins and lipid (with densities decreasing in the order listed). Use of ultracentrifugation in caesium chloride density gradients When working with nucleic acids it is important to avoid degradation due to contamination with DNases and RNases
- 12. IDENTIFICATION – (Visualization: Northern and Southern Blots) DNA and RNA molecules can be separated for analytical and preparative purposes by: Electrophoresis in agarose or polyacrylamide gels (used in sequencing, mutation detection and separation of short fragments) DNA and RNA are visualized in the gel using fluorescent dyes such as ethidium bromide. Distinct fragments can be identified based on their nucleic acid sequence with a DNA probe Complementary strands – two strands with same base pairing Denatured or separate into single strand by heating, immersion in denaturing buffers, or both Renature or rejoin by cooling or changing buffers. How its visualized? a probe with sequence matching one of the fragments can be added to single-stranded target DNA which forms a hybrid together with the target DNA fragment that has a complementary sequence (for visualization it is labelled with radioactive nucleotides (32P, 33P or 35S)…
- 13. VISUALIZATION CONT. …that can be detected by autoradiography or haptens that can be detected by staining with antibodies. Southern Blot first described by E. M. Southern To preserve the electrophoretic separation of the molecules during denaturation and hybridization, the DNA (or RNA) must be immobilized by blotting onto a membrane with high binding capacity for nucleic acids. The method for detection of specific, electrophoretically separated DNA fragments is consequently called Southern blot RNA is Northern Blot and Proteins Western Blot DNA/RNA may also be applied to membrane for detection by hybridization with probes without prior electrophoretic separation. Dot blot pipetted directly onto the membrane
- 14. SYNTHESIS plaque lift is the transfer from virus colonies to membrane Colony lift is the transfer from bacterial colonies.
- 15. Synthesis: The polymerase chain reaction PCR is used to amplify only specific segments of DNA. DNA polymerases enzyme is used to generate copies of DNA in cyclically which doubles output. DNA polymerases require a primer at the 5 side Amplified regions can be controlled by adding synthetic DNA primers. PCR takes place entirely outside living cells What is needed for PCR ? 1. Template DNA, 2. Primers, 3. The four deoxynucleotides (in triphosphate form dTTP , dCTP , dATP and dGTP), 4. DNA polymerase 5. Suitable buffer
- 16. The reaction is repeated in a cycle consisting of three steps: 1. DNA denaturation, where the reaction mix is heated to 94°C, causing separation of the two DNA template strands 2. Annealing, where the temperature is lowered so that the primers can hybridize with the template DNA. Because the primers are present in huge surplus, they will hybridize with the template before renaturation of the template DNA 3. Elongation, which is performed at the optimal working temperature of the DNA polymerase, usually around 72°C. Taq is efficient and robust, but with a high error rate as it lacks 3 to 5 exonuclease activity
- 17. Purpose of PCR In diagnostics, to identify the presence of a particular sequence (e.g. belonging to viruses or bacterial strains) in a sample. In cloning of cDNA (see below) or of fragments of genomic DNA. The pieces of DNA are first amplified by PCR, and can then be ligated into a plasmid vector. Pieces of DNA encoding parts of different proteins may be ligated together into the plasmid, for generation of chimaeric proteins. The purpose may also be construction of plasmids with novel properties. For quantification of mRNA: see reverse transcriptase (RT)–PCR, below.
- 18. ANALYSIS: DNA sequencing (i) The key element developed by Sanger and co- workers, is the use of dideoxynucleotides that when incorporated into the newly synthesized DNA strand terminate further elongation Reverse transcription and cDNA libraries (ii) Reverse transcriptases catalyse the synthesis of DNA using RNA as a template . A collection of cDNA synthesized from the whole set of isolated mRNAs is referred to as a cDNA library . cDNA libraries are therefore cell, tissue and organ specific.
- 19. Artificial chromosomes and genomic libraries Libraries can also be made from genomic DNA Yeast artificial chromosomes (YACs) permit cloning of DNA fragments exceeding a million base pairs. YACs are technically difficult to work with, and a serious disadvantage is their tendency to recombine Phage- and bacteria-derived artificial chromosomes (PACs and BACs Differences between cDNA libraries and genomic libraries cDNA libraries - are made from mRNA and will therefore vary according to cell or tissue source. Genomic libraries - are made from genomic DNA.
- 20. 6.4 PRACTICAL APPLICATIONS OF DNA/RNA TECHNOLOGY 1. Whole genome sequencing 2. Identification of novel genes 3. Analysis of gene expression 4. Semiquantitative and quantitative reverse transcriptase-polymerase chain reaction 5. Microarrays Whole Genome sequencing The assembly problems led to two different main strategies for genome sequencing 1. Human Genome Project is the hierarchical shotgun sequencing two-step method (multi step) 2. whole genome shotgun sequencing (single step)
- 21. Identification of novel genes Novel genes are primarily identified by computer analysis (in silico cloning) Two main approaches are used: 1. The first predicts the existence of genes ab initio. (without previous knowledge other than general rules for how genes are organized, and applies primarily to protein encoding genes) 2. The second is based on sequence similarities (homology) with genes already known. The input sequence can be used to search for the corresponding (orthologous) gene in a different species or for a related but different (homologous) gene in the same species. Duplicated genes in the same species are called paralogs Search tools used to identify novel genes expression are: I. Basic Local Alignment Search Tool (BLAST) program II. Homology screening is also performed in vitro, based on hybridization to DNA probes and with cDNA libraries usually ligated into bacteriophage λ-derived vectors III. expression cloning IV. subtraction cloning V. positional cloning
- 22. Analysis of gene expression What make cells different is the different selections of genes they transcribe and express Investigations of gene expression are based mainly on measurements of transcriptional activity Northern blot , is the traditional method for quantitative measurements of RNA Disadvantages 1. time consuming, 2. requires relatively large amounts of RNA and can give spurious results owing to cross-hybridization Semiquantitative and quantitative reverse transcriptase-polymerase chain reaction Disadvantages 1. The near-exponential characteristics of the amplification make it vulnerable 2. small differences in reaction conditions between tubes can have large effects 3. The number of cycles must be limited to avoid exhausting the reagents but still sufficient for reliable detection of PCR product 4. Because only the endpoint of the reaction is measured, the quantitative accuracy of the method is limited With the use of internal controls to normalize for sample to sample variations in total RNA amounts and reaction efficiency, it is possible to reach reasonable, semiquantitative estimates of specific RNA in the samples
- 23. Semiquantitative and quantitative reverse transcriptase-polymerase chain reaction Advantages 1. It does not require sophisticated apparatus, 2. and is rapid, 3. sensitive and, 4. Highly specific (depending on the appropriate choice of primers) 5. Being replaced by real-time or quantitative PCR (qPCR) (fluorescent intensity is measured) Microarrays This systematic deposition of probes is called a microarray Disadvantage 1. The electrophoretic separation of mRNA in the northern blot technique is time consuming and limits the number of samples that can be processed simultaneously Advantages 1. It yields information about the lengths of the mRNA to which the probe hybridizes 2. Ability to analyse expression of thousands of genes in a single mRNA sample.
- 24. Two-channel microarrays cDNA made from two different samples The system allows detection of upregulated or downregulated genes in the test sample versus the control, but does not give information about absolute expression levels Difference between the northern blot (or its simplified dot-blot equivalent) and the microarrays Northern Blot - monitors the expression profile of a single gene in different tissues Microarrays - the expression profile of thousands of genes in a single tissue (or compares two tissues)
- 25. FUNCTIONAL ANALYSIS OF THE ENCODED PRODUCTS Vectors for functional studies: expression vectors The vector must contain suitable promoters (expression vectors), ensuring efficient transcription in the particular cell type under study Modifying the inserted genes can be informative Gene knockdown by targeting specific mRNAs - elucidating the function of a protein when it is no longer present, or its expression is significantly reduced. • knockdown experiment, and is performed by specifically targeting the corresponding mRNAs for degradation, stopping them from being translated. • The second method is referred to as siRNA (small interference RNAs), exploiting an evolutionary Transgenic and knockout animals Techniques for genetic manipulation of experimental animals 1. Transgenic - a gene is transferred into the genome by the use of retroviral vectors or by microinjection at the zygote (fertilized egg) stage. the gene is inserted at more or less random sites in the chromosomes. The transgene is therefore heritable 2. knockout animals - are generated by experimental inactivation of one or more chromosomal genes.
- 26. IDENTIFICATION OF GENES ASSOCIATED WITH DISEASE: SINGLE NUCLEOTIDE POLYMORPHISM TYPING In medicine, identifying disease genes is of utmost value for diagnostic, therapeutic and preventive purposes. SNP - single nucleotide polymorphisms. Are alterations of single nucleotides in the genome, constitute the main source of genetic variation between two individuals of the same species International HapMap Project – used to identify SNPs inherited together Simple tandem repeats (STR) - highly useful as DNA markers in family linkage studies as well as in forensic medicine for determining paternity, clarifying alleged kinships in cases of illegal immigration and for DNA fingerprinting in criminal cases.
- 27. 6.5 PROTEIN ANALYSES The main challenges are: • the wide range of physical properties, with water solubility as a major problem • the wide range in protein concentrations, which for cellular proteins may vary with a factor of 106 (e.g. actin versus transcription factors) and for serum proteins with a factor of 109 (albumin versus cytokines) • the problem of making a comprehensive set of reporter ligands or probes for quantification by protein arrays or mapping of cell and tissue localization • determinations of three-dimensional (3D) structures, which currently cannot be predicted, but must be determined by resource-demanding techniques, mainly X-ray crystallography and nuclear magnetic resonance • the complexity of interactive networks between proteins.
- 28. Protein purification Methods for protein purification exploit • Differences in surface properties, such as distribution and accessibility of charged, polar and hydrophobic groups, which results in differences in solubility in solvents where variables are salt concentration/ionic strength, pH and content of detergents (precipitation) • Differences in size and shape (gel filtration, SDS gel electrophoresis) • Net charge (ion-exchange chromatography, isoelectric focusing) • Biological properties, mainly affinity for other molecules (affinity chromatography with natural ligand or specific antibody bound to solid phase).
- 29. Protein expression and recombinant proteins Protein expression refers to the directed synthesis of a large amount of a specific protein. The main advantage of E. coli is its simplicity in use: it is easily transformed, divides rapidly, thrives in cheap culture media and can produce large amounts of the desired protein. The disadvantage is that the protein may not be correctly folded or modified. Protein analyses by electrophoresis, western blotting and immunoprecipitation Proteins can be separated by electrophoresis. Detection of protein can be done unspecifically in the gel by staining methods Visualization of a specific protein is commonly done by transferring the proteins in the gel to a polyvinylidene fluoride (PVDF) membrane, a process known as western blotting One popular use of western blot is the identification of protein interactions
- 30. Two-dimensional electrophoresis Proteins can be separated based on their net charge. Every protein has an isoelectric point (pI), that is, a pH at which its net charge is zero Protein–protein interactions One way of identifying such interacting proteins is immunoprecipitation followed by western blot or protein sequencing Proteomics - collection of proteins that are expressed at a given time in an organism, a tissue or a cell
- 31. Expression proteomics and protein expression arrays Methods used should exhibit • high resolving power, including the ability to distinguish between modified variants of the same protein • high sensitivity • suitability for high-throughput analysis • the ability correctly to reflect relative protein concentrations. Mass spectrometry Mass spectrometry, - separates ionized molecules according to mass to charge (m/z) ratios The protein spots of interest are cut out of the 2D gel and digested with an endopeptidase, usually trypsin, which cleaves the C-terminal to the basic amino acids lysine and arginine, generating peptides of suitable lengths for further analysis
- 32. In situ mapping of proteins The main reporter ligands for in situ mapping of protein are mAbs Quantum dots are tiny semiconductors that can replace organic fluorescent dyes as reporter tags.
- 33. 6.6 BIOINFORMATICS Bioinformatics is ‘The science of managing and analysing biological data using advanced computing techniques’, and emphasizes its pivotal role in analysing genomic research data
- 34. APPLICATIONS OF BIOINFORMATICS virtual evolution, metabolic networks, molecular 3D predictions morphometrics
- 35. . UniProt (the Universal Protein Resource) is a databank dedicated to information and analysis of proteins.