Lecture RR6_Nucleic Acids 2024_2 PDF

 Course Outline Professor Rodrigo Reyes KH7: Purification detection and characterization of proteins RRL1: DNA Replication RRL2: DNA Repair and recombination RRL3: PCR and DNA sequencing RRL4: DNA cloning, experimental gene expression, int...

 Course Outline Professor Rodrigo Reyes KH7: Purification detection and characterization of proteins RRL1: DNA Replication RRL2: DNA Repair and recombination RRL3: PCR and DNA sequencing RRL4: DNA cloning, experimental gene expression, introduction to genomes KH8: Chromosomes RRL5: Genes and genomes, transposable elements RRL6: Nucleic Acids: from detection to quantification RRL7: Eukaryotic transcription RRL8: Gene regulatory sequences in RNA Pol II promoters RRL9: Proteins that regulate transcription I - General factors RRL10: Proteins that regulate transcription II – Activators RRL11: Molecular mechanisms of transcriptional activation and repression RRL12: Chromatin and epigenetics - the histone code PL1: RNA processing I 2  BIOLOGY 200 Nucleic Acids: from detection to quantification broadinstitute.org Molecular biological techniques enhance our ability to detect and analyze gene products QUALITATIVE ANALYSIS. -the nature of the molecule(s) in question. -size? -nucleotide composition? -conformation/configuration? -structure? QUANTITATIVE ANALYSIS. -determine the levels of gene products. ie…tumour markers (p53, BRCA1/2) 4  Molecular Probes can be used to find a needle in a haystack… Binding to a solid phase support membrane material : Nitrocellulose A complex mixture or of macromolecules Nylon diff DNA or RNA (seg length. , etc) molecules are exposed Southern blot - > DNA on surface of membrane other molec detect Northern blot-RNA so. can Target Detection Probe specific for Remove target recognition non-specific sequence complementary (we want a lot) ↳ Short fragment of DNA that is labelled 5 https://www.genome.gov/genetics-glossary/Southern-Blot  Single stranded oligonucleotides can be labelled using polynucleotide kinase very important * ! generating probe is Known sequence that Lys Glu Ala Phe Thr His His Gly corresponds to a gene product 5’-AAG GAG GCA TTT ACC CAT CAT GGC-3’ of interest ↳ sequence we want to detect Synthesize an oligonucleotide 5’-GCC ATG ATG GGT AAA TGC CTC CTT-3’ that has the complementary Synthesized sequence oligonucleotides do not have a phosphate at Polynucleotide Kinase (PNK) their 5’ end will phosphorylate nucleotides by transferring the  phosphate P of ATP to the free hydroxyl at the 5‘end of the 5’-GCC ATG ATG GGT AAA TGC CTC CTT-3’ oligonucleotide 6 PCR can be used to make labelled DNA probes incorporating dNTPs that carry a radiolabel on the -phosphate into PCR amplified DNA Unincorporated radioactive dNTPs substrates are removed The radiolabelled PCR product must be rendered single stranded + dGTP, dTTP, dATP, dCTP (−32P dCTP) before it can be used Fluorescence can be used instead of radioactivity GGATTCCA CTTCTGCGTTA……….CGGAGTAGAATTCCGGAAT CCTAAGGTGAAGACGCAAT……….GCCTCATCT TAAGGCCTTA 7  Analysis of nucleic acids by transferring to solid state supports (nylon, nitrocellulose) steps of southern blot : Both DNA and RNA can be separated according to size using an agarose gel. DNA is cut with a restriction enzyme and then run through an agarose gel-a diagnostic signature that reflects the DNA sequence ↳ DNA is very long mRNAs of different sizes will correspond to the various genes that encode them. he on toab direction of Both of these analytical techniques require that movementwil bring DNAIRNA the molecules be transferred to a solid state & stick to membrane support and be denatured (single stranded) for subsequent analysis.  Once covalently bound the levels and positions are permanently recorded The nucleic acids are then bound covalently to doesn't the support ↳ move This permanently records the levels (abundance) and the position (size) of the molecules following separation on the gel. The support or “blot” can then be “hybridized” with probes to any sequence that may be of interest. Washes remove non-specific signal and only complementary sequences will be detectable on the blot following autoradiography.  Why Southern, northern, western blot? Southern blot was invented by Edwin Southern, a biochemist at Oxford University, to study DNA. Later on, other scientist used similar concepts to detect specific RNA molecules (northern) and proteins (western), naming techniques in honour of Southern’s name. Ed Southern was the inventor of microarrays (lecture RR4) and studied the sequence and origin of microsatellites. Progress in science is driven by a community of scientists Invention of Southern blot, at the time, exploited three recent developments in molecular biology. Restriction enzymes Gel electrophoresis Blotting Tom Kelly and Hamilton Smith Daniel Nathans and Kathleen Danna Fred Sanger Detecting polymorphisms with probes… (Southern blots) Normal Gene Z Probe Gene X Y Gene Z Chromosome Fragment 1 Fragment 2 12 Detecting polymorphisms with probes… Mutant Gene Z Probe Gene X Y Gene Z X mutated Chromosome Fragment 1/2 13 Nucleic acid hybridization techniques and DNA detection Nucleic acid probes with complementary sequence can bind nucleic acid targets: Southern Analysis (E.M Southern) Molecular identification of RFLPs Analysis of DNA-Southern Blot Restriction Fragment Length Polymorphisms (RFLPs) Relatedness and/or Diagnostic 14 Detecting polymorphisms without probes… Non Digested EcoRI 2000 bp Amplicon 2000 Normal Gene Z Gene Z 1000 500 200 Agarose Gel stained with EtBr 2000 bp Amplicon Non Digested EcoRI 2000 Mutant Gene Z Gene Z 1000 X GAATTC -> GAATCC 500 200 15 Agarose Gel stained with EtBr Nucleic acid hybridization techniques enable RNA detection: northern analysis northern blot -RNA-gene expression Nucleic acids of complementary sequences can base pair to form double stranded hybrids, ie… Analysis of RNA-Northern Blot RNA (Heated to eliminate secondary structure) ↳ so migration only depends on length Tissue-specific expression Denaturing electrophoresis in buffer Stage-specific containing formaldehyde to block formation of secondary structure (temporal) expression 16 Nucleic acid directed sequence recognition is the basis of CRISPR Cas Similar to Southern blot, northern blot and microarrays, CRISPR-Cas uses a short RNA molecule to direct the search of a specific sequence in the genome. The Cas9 protein is an endonuclease, that will cut the two strands once the sequence has been found. 17 Using Quantitative RT-PCR (RT-qPCR) to determine mRNA levels RT reaction will convert all mRNA to cDNA. This reaction can then be subjected to a PCR reaction that includes an intercalating fluorescent dye that emits signal when incorporated into the growing DNA polymer. The more DNA produced; the more fluorescence signal is detectable. This can be measured in fluorescent when binding real time. to dsDNA 18 Using Quantitative RT-PCR (RT-qPCR) to determine mRNA levels All PCR reactions go through an exponential phase, a linear phase, and finally they reach a plateau. This plateau is reached much faster (in fewer cycles) in samples with greater amounts of starting material (cDNA). The amount of starting material can be calculated by monitoring the number of cycles it takes to reach the plateau phase. The more cDNA in the original sample the faster the plateau is achieved. This is directly proportional to the mRNA abundance in the original sample 19 RNA-seq: a method for studying global gene expression understanding levels of mRNA 20 RNA-seq: a method for studying global gene expression 21 RNA-seq: a method for studying global gene expression ↳ next gen, sequencing 22 RNA-seq: a method for studying global gene expression 23 Next generation sequencing techniques, such as Illumina sequencing, do a PCR in a solid support to obtain lots of copies of the same molecule in a very small space. They use these DNA clusters for sequencing From lecture RR3 24  Cluster analysis can identify coordinately regulated genes mostly in larrae similarily expressed mostly in adults Agriotes sputator (Wireworm, affecting potato production) https://insektarium.net/ https://doi.org/10.1038/s41598-024-74495-1 Recap Analysis of nucleic acids (DNA and RNA) can be done through the immobilization of molecules on a solid phase and the use of specific probes Southern blot (DNA) is often used to detect polymorphisms Northern blot (RNA) is used to study gene expression Quantitative RT-qPCR estimates absolute numbers of mRNA molecules in a sample RNA-sequencing is used to obtain a picture of global gene expression in a sample  Course Outline Professor Rodrigo Reyes KH7: Purification detection and characterization of proteins RRL1: DNA Replication RRL2: DNA Repair and recombination RRL3: PCR and DNA sequencing RRL4: DNA cloning, experimental gene expression, introduction to genomes KH8: Chromosomes RRL5: Genes and genomes, transposable elements RRL6: Nucleic Acids: from detection to quantification RRL7: Eukaryotic transcription RRL8: Gene regulatory sequences in RNA Pol II promoters RRL9: Proteins that regulate transcription I - General factors RRL10: Proteins that regulate transcription II – Activators RRL11: Molecular mechanisms of transcriptional activation and repression RRL12: Chromatin and epigenetics - the histone code PL1: RNA processing I 27