2022 Chem 161b Analysis of Nucleic Acids PDF

Analysis of Nucleic Acids Purification and detection of nucleic acids l DNA can be isolated directly from living, dead and even extinct species. l Two different types of DNA can be isolated: genomic DNA (gDNA) and complementary DNA (cDNA) l Genomic DNA – chromo...

Analysis of Nucleic Acids Purification and detection of nucleic acids l DNA can be isolated directly from living, dead and even extinct species. l Two different types of DNA can be isolated: genomic DNA (gDNA) and complementary DNA (cDNA) l Genomic DNA – chromosomal DNA, most organisms have the same genomic DNA in every cell l Complementary DNA – synthesized from a single stranded RNA template in a reaction catalyzed by reverse 54 transcriptase. Figure 5.1. Genomic DNA (gDNA) vs Complimentary DNA (cDNA). Lefthand Diagram shows the processing of genomic DNA within a cell to produce a protein (Upper blue panel shows the structural elements common to eukaryotic genes. The process of gene transcription produces a messenger RNA (mRNA) molecule that must be modified post- translationally, gray panel, to remove the non-coding intron sequences and add the 5′- CAP and Poly-A-Tail sections. The mature mRNA is transported from the nucleus to the 55 cytoplasm where it is translated by the ribosome into the protein sequence, red panel.) Righthand Diagram shows that the isolation of mRNA from a cell can be used to synthesize cDNA using the enzyme reverse transcriptase. The resulting cDNA only contains elements from the mature mRNA, including the exons and poly-A tail. Image modified from Wikipedia DNA isolation techniques l The first isolation was done in 1869 by Friedrich Miescher. l Now purification kits are available from multiple manufacturers. Scheme for DNA isolation and purification l Collect cells l Cell lysis (detergent and surfactants, protease, RNase) l Treat with concentrated salt solution to clump debris 56 (proteins, lipids and RNA) l Centrifuge to separate clump from DNA solution  DNA purification l Ethanol precipitation – ice-cold ethanol or isopropanol. DNA will aggregate and forms a pellet upon centrifugation. Improved by increasing ionic strength by adding sodium acetate. l Phenol-chloroform – phenol denatures proteins, after centrifugation, denatured proteins stay in the organic phase, while Figure 5.2 Silica Spin Column Used for DNA Purification. Spin column-based nucleic acid aqueous phase containing nucleic purification is a solid phase extraction method to acid is mixed with chloroform that quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid removes phenol from the residue. phase of silica under certain conditions and then released when those conditions are altered. For l Minicolumn purification – relies binding, a buffer solution is added to the DNA lysate along with ethanol or isopropanol. This on the fact that the nucleic acids forms the binding solution. The binding solution may bind to the solid phase (e.g. is transferred to a spin column and the column is put in a centrifuge. silica) depending on the pH and the Image by Squidonius salt concentration of the buffer. Gel electrophoresis of nucleic acids A solution of DNA is colorless, and except for being viscous at high concentrations, is visually indistinguishable from water. Gel electrophoresis have been developed to detect and analyze DNA. A solution of DNA is deposited at one end of a gel slab. This gel is made from polymers such as agarose, which is a polysaccharide isolated from seaweed. The DNA is then forced through the gel by an electrical current, with DNA molecules moving toward the positive electrode. 62 DNA fingerprinting l A technique is used to identify patterns that occur in DNA. No two organisms have identical DNA so this procedure can be used to identify if a sample of DNA came from a particular individual. l Examples : l Identification of whether a sample of DNA found at a crime scene belongs to one of three suspects. l It can be used to identify whether individuals carry genes for certain genetic diseases. 63 Restriction fragment length polymorphism (RFLP) l DNA from differing sources will have variations or polymorphisms throughout the sequence. l Using restriction enzymes, these differences in sequences may be teased out. However, if the entirety of the human genome is treated with a restriction enzyme, many indecipherable fragments would be made. The resulting agarose gel would simply show a large smear of DNA. l RFLP analysis requires that a probe to a specific area of DNA be used to identify specific locations. Agarose gels would be transferred to a membrane or filter where they would be hybridized to these radioactive probes. Restriction endonucleases - Enzymes that catalyze cleavage of DNA at selective sites - Provides a reproducible method for opening (linearizing ) circular vectors.  Since people are 2N, they have pairs of homologous chromosomes with the same loci. However, these loci may contain different alleles. The phenotype for these alleles may or may not contain restriction sites. The presence or absence of a restriction site may arise from single nucleotide polymorphisms (SNPs) that reveal the natural Homologous chromosomes with restriction sites noted by triangles. the variation between people. rectangle sitting on the chromosomes correspond to a probe locus. Credit: Jeremy Seto (CC0) 66 Following restriction digestion, the samples are resolved on an agarose gel. Digestion of genomic DNA will result in a large smear. Following transfer of the DNA onto a membrane through capillary action, the membrane is probed with radioactive probe DNA. The probe binds selectively to complementary sequences to reveal a series of distinct bands. An interactive demonstration of the first DNA fingerprinting. Credit: Oder Zeichner: abigail [ or CC-BY-SA-3.0] /Autoradiogram  BIOTECHNOLOGY Biotechnology – application of organisms, biological cells, cell components and biological processes to practical operations and problem solving. Examples: 1. Making of food commodities, e.g. cheese, wine. 2. Use of bacterial cells to produce larger quantities of scarce proteins needed to treat diseases. 3. Use of enzymes in industrial production of specialty chemicals. 4. Modify genes of a plant so it can grow under adverse conditions. 5. Production of fuel alcohols from plant starch. 6. Using bacteria and plants for cleanup of chemical wastes. 7. Mining of metals, “bioleaching” – use of microorganisms to extract metals. 8. Gene replacement in individuals with genetic disorders. 9. Identification of biological specimens at crime scenes. 10. Use recombinant proteins/ hormones for production of animals and animal products. The advances in biotechnology depend on our ability to change and manipulate the genetic characteristics of fundamental life forms. DNA Sequencing 1. Maxam-Gilbert method – based on chemical modification of DNA and subsequent cleavage at specific bases. -Labeling at 5’ end with 32P-nucleotide -Chemical modification of bases and cleavage - Electrophoresis and autoradiography 2. Sanger chain termination method – use of ddNTPs to terminate DNA replication, producing varying lengths of DNA that are analyzed by electrophoresis and autoradiography. - Requirements: template (3’à5’), short primer (5’à3’), DNA polymerase, dNTPs (labeled dATP, dGTP, dCTP, dTTP), ddNTPs  Single stranded DNA, with labeled 5’ end G – methylation by dimethyl sulfate A and G – depurination by formic acid C and T – hydrolysis by hydrazine C – hydrazine + 5M NaCl Piperidine – cleavage of phosphodiester bond next to the modified base S Sanger sequencing  Polymerase chain reaction - Method used to synthesize amplified quantities of specific sequences of DNA from small amounts of DNA using heat stable DNA polymerase - Conceived by Kary Mullis in 1983 (Nobel prize in Chemistry 1993). Components of a PCR reaction mixture: 1. DNA Template– The DNA of interest from the sample. 2. DNA Polymerase– Taq Polymerase is used. It is thermostable and does not denature at very high temperatures. 3. Oligonucleotide Primers- These are the short stretches of single- stranded DNA complementary to the 3’ ends of sense and anti- sense strands. 4. Deoxyribonucleotide triphosphate– These provide energy for polymerization and are the building blocks for the synthesis of DNA. These are single units of bases. 5. Buffer System– Magnesium and Potassium provide optimum conditions for DNA denaturation and renaturation. It is also important for fidelity, polymerase activity, and stability.  Recombinant DNA technology I. Molecular cloning (DNA recombination) - Covalent insertion of a DNA fragment from one type of cell or organism into the replicating DNA of another cell. - Many copies of the gene and the translated protein are produced in the host cell Steps in molecular cloning 1. Select and isolate a DNA molecule to serve as the carrier for the foreign DNA. Vectors 2. Cleave the DNA strands of the vector with a restriction endonuclease. 3. Prepare the foreign DNA, treat with same restriction endonuclease and then insert the foreign DNA into the vector (ligation). This produces the recombinant DNA. 4. Introduce recombinant DNA into host organism (often bacteria, or animal or plant cell). (Transformation). 5. Identify and screen the host cells for the ones which accepted and are replicating the recombinant DNA  Cloning vectors 1. Plasmids – self-replicating, extrachromosomal DNA molecules found in bacterial cells. - closed circular, double-stranded DNA, 3000-30,000 base pairs - Bacterial plasmids normally contain genes for proteins that confer specialized/protective functions on the organism, e.g. enzymes for degrading toxins and antibiotics. 2. Bacteriophage DNA – viral DNA, used to “infect” a host cell, double stranded ~50,000 base pairs, useful for larger DNA fragments ~23,000 bp. An example of a plasmid for cloning with E.coli is pUC18 Ori – origin of replication DNA sequence of 50 – 100 base pairs that must be present in a plasmid for it to replicate. Host-cell enzymes bind to ORI, initiating replication of the circular plasmid. Selectable gene, most commonly a drug-resistance gene encoding an enzyme that inactivates a specific antibiotic. Example: the ampicillin-resistance gene (ampr) encodes β-lactamase, which inactivates the antibiotic ampicillin. lacZ – part of the lac operon codes for beta-galactosidase Useful for screening of transformants Blue-white screening X-gal – substrate for beta-galactosidase X-gal, BCIG for 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside galactose linked to a substituted indole. 5,5'-dibromo-4,4'-dichloro- indigo, BLUE PRODUCT Preparation of foreign DNA 1. Chemical synthesis 2. Reverse transcription of mRNA Uses the reverse transcriptase (RT), enzyme used to generate complementary DNA (cDNA) from an RNA template. This enzyme is mainly associated with retroviruses, which uses the process to produce cDNA that can integrate in the host cell genome, thus replicating and infecting the cell. 3. Restriction endonuclease action on larger DNA fragment REVERSE TRANSCRIPTION Restriction endonucleases - Enzymes that catalyze cleavage of DNA at selective sites - Provides a reproducible method for opening (linearizing ) circular vectors.  Ligation - The method of covalent joining of foreign DNA to the vector ends and final closure of the circular DNA - Enzyme: DNA ligase Transformation - Introduction of foreign DNA into host cells. - E.g. E. coli as host cells - The addition of CaCl2 to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged Ca2+ ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. - The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time  Selection - Successful transformants are selected using antibiotic resistance. - Selectable gene, most commonly a drug-resistance gene encoding an enzyme that inactivates a specific antibiotic. - Example: the ampicillin-resistance gene (ampr) encodes β-lactamase, which inactivates the antibiotic ampicillin.  An example of a plasmid for cloning with E.coli is pUC18 lacZ – part of the lac operon codes for beta-galactosidase Useful for screening of transformants Blue-white screening Blue – with active beta- galactosidase White – beta galactosidase not expressed X-gal – substrate for beta- galactosidase X-gal, BCIG for 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside galactose linked to a substituted indole. 5,5'-dibromo-4,4'-dichloro- indigo, BLUE PRODUCT