Lecture 14 Nucleic Acid Analysis MCQ PDF

Basics of Nucleic Acid Analysis 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). Lear...

Basics of Nucleic Acid Analysis 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). Learning Outcomes Describe principal steps and science for isolating and purifying nucleic acids Explain the principles underlying basic nucleic acid analysis techniques Give examples of use in bioscience research Give examples of use in medicine Give examples of use in forensics Nucleic Acids How to obtain nucleic acids Techniques to analyse nucleic acids How to manipulate nucleic acids Types of data obtained and interpretation thereof Why do we want to? DNA RNA PROTEIN Where do we obtain the nucleic acid sample? Bone marrow Faeces Mitochondria Sewerage Blood Soil CSF Water Saliva Air Nasal fluid Your coffee cup? Applications of Nucleic Acid Analysis Genes and genome organization Gene expression and function Research Evolutionary relationships Medicine Regulation of gene expression Pathologies determination Forensic science Diagnosis and treatment Archaeology Screening and testing Agriculture Paternity testing Identification Biotechnology Barcoding Modifying organisms Extracting and Purifying Nucleic Acids Modern methods based on DNA or RNA binding to silica matrix Allows kits and automation Negatively charged silica Positively charged DNA or RNA Why is the DNA / RNA positively charged here?  Extracting and Purifying Nucleic Acids Aim is to: 1) Release nucleic acid from cell into conditions which will not destroy it. 2) Remove contaminants. 3) Provide conditions which allow binding to silica matrix. 4) Remove rest of contaminants. 5) Change conditions so nucleic acids no longer binds to matrix. 6) Then collect nucleic acid in a solution which will not destroy it and allow it to be stored or analysed. What destroys nucleic acids?  Preparation of samples Need to collect and break open the cells By grinding, homogenizing or enzymes e.g. lysozyme Plant tissues, fungi and bacteria need to break the cell wall Mammalian cells just need to disrupt membrane (lyse)  Extracting the nucleic acids Bind DNA Change buffer and elute DNA Break open cells Precipitate out proteins Knowledge of pH, ionic Wash DNA and remove charges, salts, contaminants molecular charges and interactions. Re-suspend DNA to correct concentration  Isolation of messenger RNA (mRNA) Gene expression studies isolate mRNA. Why? RNA less stable than DNA Many molecular biology techniques work only with DNA Isolated mRNA converted to complementary DNA (cDNA) cDNA is more stable Can manipulate cDNA more easily cDNA can be a template for PCR or sequenced Need oligonucleotide primer and enzyme, Reverse Transcriptase  Reverse transcription - making DNA from RNA Eukaryotic mRNA has a polyA tail –AAAAAAAAAAAAA To convert isolated mRNA to cDNA need TTTTTTTTTTTTT oligonucleotide primer and Reverse Transcriptase enzyme Remember NORMAL PROCESS is: DNA RNA PROTEIN Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Initial Analysis of Nucleic Acid: Spectroscopy To determine concentration and purity of nucleic acid sample UV-Vis Spectrophotometer NanoDrop  Nucleic Acid Spectroscopy DNA and RNA maximally absorb light at λ = 260 nm (UV range) DNA concentration measured by: Abs260nm x 50 x dilution factor = μg/mL DNA Absorptivity constant is 50 for DNA and 40 for RNA (μg/mL) Proteins will maximally absorb light at λ = 280 nm (UV range) DNA purity is determined by the absorbance ratio of A260/A280 Ratio of 1.8 - 2 is pure; ratio 2.1 other organic contamination e.g. RNA, phenols DNA Manipulation: PCR and amplification Polymerase Chain Reaction: Exponential amplification of DNA Revolutionised molecular biology Developed in 1980s Kary Mullis won Nobel Prize for Chemistry in 1993 https://www.nobelprize.org/prizes/chemistry/1993/mullis/facts/ Link to short video (3 min 31 sec) : PCR - Polymerase Chain Reaction - Simple Animated Tutorial https://www.youtube.com/watch?v=DkT6XHWne6E  https://www.thermofisher.com/.../pcr-basics.html PCR Amplification of DNA Need: Template DNA; Specific oligonucleotide primers; Taq DNA polymerase; Correct buffers; PCR machine. Introducing specific mutations by PCR Site-directed mutagenesis Design an oligonucleotide with KNOWN and SPECIFIC change in bases Mutated oligonucleotide used in PCR - increase amount mutated DNA Will change amino acid sequence in PREDICTIVE manner e.g. change Tyrosine (codon TAC) to Alanine (codon GCC) Original techniques developed by Michael Smith Won the Nobel Prize in Chemistry in 1993 https://www.nobelprize.org/prizes/chemistry/1993/smith/facts/  Analysing DNA by Electrophoresis a) Checking DNA is present and of correct size b) Checking DNA sequence is correct c) Checking if mutations present – wanted or unwanted… Agarose gel Capillary electrophoresis (DNA sequencing) Used for: (a), (c) sometimes* Used for (b) and (c) always Capillary Electrophoresis: DNA Sequencing Standard for DNA sequencing DNA molecules move in an electrical field Electrophoretic mobility due to size Detection by UV or fluorescent tags Data interpreted by computer  DNA sequencing: (Sanger Sequencing) Walter Gilbert and Frederick Sanger won Nobel Prize in Chemistry 1980 https://www.nobelprize.org/ prizes/chemistry/1980/summary/ Agarose Gel Electrophoresis Separation by size through a molecular mesh DNA molecules move in an electrical field Different gel percentages of agarose in buffer (grams of agarose per 100 mL buffer; a weight per volume) Used to resolve differently sized DNA bands Restriction Enzyme (RE) Mapping REs cut DNA in specific places Differently sized DNA fragments made DNA separated by size on an agarose gel Size determined by comparing to a DNA ladder (fragments of known size) Done to confirm success of inserting DNA fragments into plasmid To check known DNA plasmid constructs Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Hybridization Techniques Used for gene expression analysis or for identifying genetic variations (mutations) within individual genomes. DNA Microarrays Current; for DNA and RNA analysis Southern Blots Old; for DNA analysis Northern Blots Old; for RNA analysis https://www.genome.gov/about-genomics/fact-sheets/DNA-Microarray-Technology  DNA Microarray Chips Short oligonucleotides are printed onto chip Know oligo sequence at each spot Many, many sequences per chip If gene expression study: isolate mRNA, converted to cDNA, RE cut and PCR amplify If genomic variation study; isolate gDNA, RE cut and PCR amplify For both DNA fragments tagged with fluorescent label YouTube video from HHMI https://www.youtube.com/watch?v=ui4BOtwJEXs Comparing different tissues to see what genes are expressed  Chips analysed by scanner and computer Can compare relative gene expression e.g. Normal tissue cDNA labelled with red and cancer tissue cDNA with green Interpretation by intensities and colour overlap www.genomicseducation.ca https://www.youtube.com/watch?v=ePFE7yg7LvM Applications of Nucleic Acid Analysis Genes and genome organization Gene expression and function Research Evolutionary relationships Medicine Regulation of gene expression Pathologies determination Forensic science Diagnosis and treatment Archaeology Screening and testing Agriculture Paternity testing Identification Biotechnology Barcoding Modifying organisms Applications in Bioscience Research: Examining functional organization of genes Examining regulation of gene expression Identifying key regulatory genes in development. Genetic influences on cancer or aging or metabolism Evolutionary relationships between organisms Identification of human and hominid prehistory and relationships Species identification (bar-coding) Applications in Medicine Genetic Testing for a known mutation Prevention Done on targeted population level e.g. BRCA genes or haemophilia Diagnosis Requires specific gene / protein to be Treatment affected Epidemiology Tracking prevalence of disease (pathogen) in population Forensics applications Short tandem repeat profiling (DNA fingerprinting) Familial relationship analysis Identification of human remains Monitoring and tracking endangered species (DNA barcoding) - preventing illegal trade Applications of Nucleic Acid Analysis Genetic analysis (genotype) in forms functional (phenotype) analysis Techniques and technology are interdependent Technology is rapidly evolving: - new methods and applications are being developed Useful analysis of the data reliant on computing and bio- informatics Ethical and safety issues always need be considered before developing and using recombinant DNA technology  Further Reading Fun Reading A Brief History of Everyone Who Ever Lived by Adam Rutherford Fun Singalong? PCR song from BioRad https://www.youtube.com/watch?v=FpxwJNNufko Chapter 17 Biotechnology Smartbook assignment Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). MCQ quiz for Lecture 14: Nucleic Acids Analysis Answers will be given in your Seminar sessions – with further discussion. You must attempt before your seminar session. These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster Q1) Which of the following statements is incorrect? a) The Central Dogma of modern biology is DNA to RNA to Protein. b) The correct concentration of ionic salts and buffer pH is important for DNA purification. c) Analysis of DNA sequences is used to determine protein sequences. d) DNA can be made from RNA. e) DNA sequence information is obtained via polymerase chain reaction. Q2) Which of the following are DNA Microarray chips not used for? a) Indirectly analysing what proteins maybe expressed in a chosen tissue of interest. b) Analysing if there is genomic variation between tissue samples. c) Directly quantifying the amount of DNA in a sample. d) Comparing differences in gene expression between normal and pathophysiological tissue samples. e) Allow indirect analysis of mRNA present in a cell. Q3) For agarose gel electrophoresis which of the given statements are incorrect? a) The DNA molecule is separated by its size (number of base pairs). b) A 1 % (w/v) agarose gel means 1g agarose in 100mL water, melted and cast. c) The DNA moves via an electric current. d) The size of a DNA band is measured by comparison to a ‘ladder’ of known band sizes. e) The DNA molecules moves through the gel towards the anode. Q4) Your UG project students need to measure the concentration of their purified DNA sample. The technique they need to use is? a) UV Spectroscopy b) DNA capillary sequencing c) Agarose gel electrophoresis d) Microchip hybridisation e) Polymerase chain reaction Q5) Nucleic acid analysis is useful for? a) Examining evolutionary relationships between species. b) Tracking pathogen spread. c) Monitoring endangered species and the wildlife trade. d) Searching for cancer causing mutations. e) All of the above.