Genetics Questions - Molecular Genetic Analysis PDF
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This document provides an outline of molecular genetic analysis and biotechnology techniques, beginning with restriction enzymes and Polymerase Chain Reaction (PCR), and concluding with recombinant DNA technology and gene cloning. The techniques are described in detail, offering explanations and key features.
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2171 Molecular Genetic Analysis and Biotechnology part 1 - Explain the different molecular genetics techniques - Restriction enzymes - Used by bacteria to defend against viruses - Endonucleases that cut DNA at spec...
2171 Molecular Genetic Analysis and Biotechnology part 1 - Explain the different molecular genetics techniques - Restriction enzymes - Used by bacteria to defend against viruses - Endonucleases that cut DNA at specific nucleotide sequences (restriction sites) - Type II are used in molecular genetics → recognize short palindromic sequences and make double stranded cuts, producing cohesive (sticky) or blunt ends - Restriction fragment length polymorphism (RFLPs) - Uses restriction enzymes and gel electrophoresis - Analyzes variations (polymorphisms) in homologous DNA sequences - Probes (labeled DNA or RNA molecules with a base sequence complementary to the sequence of interest) can be used to visualize fragments - Gel electrophoresis - Separates molecules based on size and electrical charge - DNA (negative) moves toward positive electrode when current is applied - Smaller fragments move faster - Southern Blotting - Transfers DNA fragments from a gel to solid medium - Allows DNA sequences to be further analyzed - Polymerase Chain reaction (PCR) - In vitro method for quickly amplifying DNA fragments (each cycle doubles the target sequence) - Uses a heat-stable DNA polymerase, primers that flank the target sequence, and deoxynucleotide triphosphates (dNTPs) - Involves repeated cycles of denaturation (seperating strands by heating), annealing (allowing primers to bind to target sequence), and extension (DNA polymerase synthesizing new strands from the primers) - Quantitative PCR (qPCR) AKA real-time PCR - More precise quantification of DNA - Uses fluorescence to track the amplification in real-time - Measurements taken at the end of the cycle to create amplification curve - Cycle threshold (ct) value used for quantification - Cycle number in which amplification exceeds a set threshold - Lower value indicates higher starting amount of DNA - Useful for analyzing copy number variations (CNVs) → duplications or deletions of particular segments - Determines if a sample DNA contains more or less target sections than a reference gene - Recombinant DNA Technology - Aka genetic engineering - Manipulate DNA from multiple sources - Analyze, alter and recombine DNA - Gene cloning - Ampllifies a specific DNA fragment using bacteria: - Plasmids: circular DNA molecules in bacteria that replicate independently of the bacterial chromosome - Cloning vectors: altered plasmids designed to insert foreign DNA - Transformation: inserting the recombinant plasmid into bacteria - Selection markers: genes that provide a selectable trait, which helps identify bacteria that have taken up the plasmid - Expression vectors - Specialized cloning vectors - Make it easier for the inserted foreign DNA to be transcribed and translated inside the target organism - Contain regulatory elements that the host’s machinery recognizes - enerate a restriction map G - - Interpret/predict results of restriction map, PCR, qPCR or Southern blot analysis - Restriction map - diagram showing the locations of restriction enzyme sites within a DNA molecule - PCR - Presence of band → successful amplification of the target sequence - bsence of band → target sequence not in the sample, or problems in A PCR - Size of band → should match the expected size based on location of the primers on the target sequence. - Deviations → insertions, deletions, etc - Intensity of the band → estimate of how much target sequence there is - qPCR (quantitative, measures amount in sample) - CT value → inversely proportional to amount of target DNA in the sample - Use a reference gene - Southern blot analysis (detect specific sequences) - Presence of band → presence of target sequence - Size of band → size of fragment containing target sequence - Intensity of band → rough estimate of how much target sequence is present 2171 Molecular Genetic Analysis and Biotechnology part 2 - Outline the different sequencing technologies and their key features - Dideoxy Sequencing (Sanger Sequencing) - Uses dideoxynucleoside triphosphate (ddNTP) - lacks the 3’-OH group required for DNA synthesis - When incorporated into a growing DNA strand during synthesis, synthesis stops - Reaction mixture contains: - Template DNA - A primer - DNA polymerase - dNTPs - Small amount of one type of ddNTP - DNA polymerase incorporates either dNTPs or ddNTPs (halt) - Various fragments of different lengths (stops at ddNTP) - DNA fragments separated due to gel electrophoresis - Next generation sequencing (NGS) - Sequence millions of DNA fragments simultaneously - Rapid and cost-effective sequencing of genomes - Relies on sequencing by synthesis (similar to dideoxy) - Steps: - Fragmentation: target DNA fragmented into small overlapping fragments - Adaptor ligation: adaptors added to ends of fragments - Immobilization and amplification: immobilized on a solid surface, then amplified to generate clusters of identical DNA molecules - equencing by synthesis: synthesizing a complementary strand S using fluorescently labeled nucleotides - Illumina sequencing (NGS platform) - NGS generates shorter reads than third-generation sequencing - Third-generation sequencing - Can sequence longer fragments than NGS - PacBio sequencing(example of third generation sequencing) - Uses adaptors for immobilization and primer annealing - Sequences longer fragments than NGS - Nanopore sequencing(another example) - Exception to sequencing by synthesis approach - Uses adaptor protein to pass single DNA molecule through a nanopore (tiny hole in membrane) - Changes in electrical current are used to determine the sequence - Higher error rate than PacBio or NGS - Resolve a dideoxysequencing gel - Reading the gel: - Gel is read from bottom to top - Smallest fragments at bottom - Bottom is 5’ end - Lanes correspond to different ddNTP (ddATP, ddGTP…) - Outline the main steps in genome editing - 1. Selection of molecular scissors - Meganucleases - Naturally occurring enzymes - Recognize long (15-40bp) target sequences - Highly specific, but limited target flexibility - ZFNs - Artificial restriction enzymes - Combine a zinc finger DNA-binding domain with a DNA cleavage domain - Off target effects are a concern - Require multiple zinc finger domains/proteins and the cleavage site is not specific - TALENs - Similar to ZFNs, but with more easily extendable DNA-binding motif - Higher specificity - Prone to off-target effects, labour-intensive to construct/design - CRISPR/Cas9 - Uses single guide RNA (sgRNA) with a Cas9 nuclease - sgRNA target a specific sequence, Cas9 creates a double strand break at target - owerful, but off target cleavage is possible as Cas9 tolerates P some mismatches - Also requires a protospacer adjacent motif (PAM) - Argonaute - Alternative to CRISPR-Cas - Uses short interfering RNA (siRNA) or microRNA as guides in eukaryotes - Uses DNA fragments as guides in archaea - 2. Target site and guide selection - For CRISPR-Cas and argonaute, a guide must be selected (sgRNA,siRNA,microRNA) - 3. DNA cleavage and repair - The molecular scissors create a DSB in the target site, and the cell’s DNA repair mechanisms attempt to repair it - Two main pathways: - Non homologous end joining (NHEJ) - Small insertions or deletions at the break site, potentially disrupting the gene function - Homology-directed repair (HDR) - Uses a provided DNA template to repair the DSB - Less efficient than NHEJ - Explain the key features, similarities and differences, and limitations of the molecular scissors - Similarities: - All create DSBs in DNA - Reliance on cellular repair mechanisms (NHEJ or HDR) - Knockouts, insertion, correction - Differences - Origin - Meganucleases → naturally occurring - ZFNs, TALENs, and CRISPR-Cas9 systems → engineered - Target recognition mechanism - Meganucleases → bind directly to long sequences - ZFNs and TALENs → use engineered protein domains - CRISPR → DNA/RNA hybridization - Argonaute → small DNA or RNA guides - Ease of design - Crispr is easiest to design Molecular Genetic Analysis and Biotechnology part 3 - Explain the difference between forward and reverse genetics - wo main molecular genetics approaches for studying genes: forward and T reverse genetics Feature Forward Genetics Reverse geentics Starting point pheotype gene Goal Identify the gene causing the nderstand the gene’s U phenotype function methods andom mutagnesis, R argeted gene alteration, T phenotypic screening phenotypic analysis - Reverse genetics & targeted alteration of gene expression - Gene knockout - Inactivating or removal/replacal or the gene - Gene knockdown - Reducing gene expression through techniques like RNA interference (RNAi) - Gene overexpression - Increasing gene expression, often by modifying the promoter - Ectopic gene expression - Expressing the gene in a location or time where it is not usually active - Reverse genetics often uses transgenic organisms - organisms whose genomes have been permanently modified by the introduction of a transgene (an engineered piece of DNA) - If a transgene is integrated into the organism's germline cells, it can be passed onto future generations. - Forward genetics - Random mutagenesis - Exposing an organism to mutagens (chemically or radiation) to induce random mutations - Phenotypic screening - After mutagenesis, find those exhibiting desired mutant phenotype - Explain the different strategies for studying gene functions - See above - Define the key terms - See above Genomics and proteomics - Describe the steps in map-based and whole-genome shotgun sequencing - These are the two main approach for sequencing entire genomes - Map-based sequencing (time consuming and labour intensive) - Genetic maps (linkage maps)→ show the relative order of genes and genetic markers based on recombination frequencies during meiosis - Physical maps (more accurate and higher resolution) → based on direct analysis of DNA and provide distances between genetic markers (bp, kbp, mbp) - Steps in map-based sequencing: - Creating a genetic map - Creating a physical map - Selecting clones (overlapping DNA fragments) for sequencing - Sequencing the clones - Whole-genome shotgun sequencing (faster, less expensive) - Steps: - DNA fragmentation - Cloning and sequencing - Sequence assembly - Explain haplotype, linkage disequilibrium and genome-wide association studies - Haplotype - The specific set of SNPs and other genetic variants observed on a single chromosome - Combination of alleles at different loci inherited together from one parent - SNPs within a haplotype are physically linked and tend to be inherited together - Recombination can shuffle them leading to new haplotypes - Linkage disequilibrium (LD) - Non-random association of alleles at different gene loci - Certain allele combinations at different loci occur together more/less frequently than expected by chance if they were independently assorted - When alleles are always sorted independently, they are in linkage equilibrium - Mutation and recombination influence LD - Genome-wide association studies (GWAS) - Aim to identify associations between genetic regions and traits - Tag-SNPs → representative SNP in a genomic region with a non-random collection of SNPs defining a hpalotype - GWAS identify common variants that tag a region of linkage disequilibrium containing the potentialcausal variant(s), which are the ctual mutations causing the trait. The tag-SNPs themselves might not be a the causal variants but are in linkage disequilibrium with them. - Explain the difference between homologous, orthologous, and paralogous genes - Homologous genes (genes that are evolutionarily related) - Two types: - Orthologs - Found in different species - Evolved from the same gene in a common ancestor - Usually retain the same biochemical function and specificity - Paralogs - Found in the same species - Arise from gene duplication events - Usually retain the same biochemical function but have a different specificity - Describe the steps in the methods used in transcriptomics and proteomics - Transcriptomics - Study of all RNA molecules found in an organism or tissue - Coding (mRNA) and non-coding RNA (tRNA, rRNA, etc) - Not all mRNA molecules are translated into proteins - Key aspects: - Reliance on genome sequence - Reverse transcriptase (RNA→DNA) - Two techniques: - RNA sequencing - Microarray analysis - Proteomics - Studying the proteome (all proteins present in the cell/tissue/organism) - Technique: - Mass spectrometry - Genomics → provides the foundation by revealing the blueprint of life encoded in the DNA sequence. - Transcriptomics → examines the dynamic process of gene expression, providing insights into which genes are actively being transcribed into RNA. - Proteomics → investigates the final products of gene expression, the proteins, which carry out the diverse functions that determine an organism's traits and responses to its environment. Epigenetics - xplain the similarities and differences between the epigenetics modifications and their E effect on gene expression - pigenetics → heritable changes in gene expression that occur without E alterations to the DNA sequence - Types of modifications: - DNA methylation - Addition of a methyl group to a cytosine base - Typically occurs at CpG islands (regions of DNA rich in cytosine and guanine nucleotides) - Makes DNA less accessible by transcription machinery Repressing gene expression: - Interfere with the binding of transcription factors - Recruit proteins that compact chromatin - Histone modifications - Histones are proteins around which DNA wraps to form chromatin - Types of modifications: - Acetylation - Loosens chromatin structure(increased gene expression) - Methylation - Phosphorylation - Ubiquitination - Non-coding RNAs - Can interact with mRNA molecules or by recruit chromatin-modifying complexes, regulating gene expression - Types: - miRNAs (microRNAs) - Leads to mRNA degradation or translational repression - lncRNAs (long non-coding RNAs) - acting as scaffolds for protein complexes - guiding chromatin-modifying enzymes to specific genomic locations - directly interacting with transcription factors - Explain the effects of epigenetic modifications on DNA, RNA, histone andchromatin - See above - Outline the different writers, readers and erasers and their general functions - Writers - Establish epigenetic code by adding modifications to DNA and Histones - Enzymes such as histone acetyltransferases (HATs) and histone methyltransferases (HMTs) add modifications to histone tails. - Readers - Interpret epigenetic code, recruiting other proteins or complexes to activate or repress gene expression - roteins containing specific domains, such as bromodomains and P chromodomains, recognize and bind to particular histone modifications - Erasers - Remove modifications - Outline the histone code - Active gene expression - acetylation of specific lysine residues on histone tails - Gene repression - methylation of particular lysine or arginine residues Population Genetics - List the assumptions and predictions of the Hardy-Weinberg law - Assumptions: - Large population size - Non-random mating - No migration - No mutation - No natural selection - Predictions: - Constant allele frequencies - Stable genotypic frequencies - Calculate the allele and genotype frequencies - Genotype frequency = (Number of individuals with a particular genotype) / (Total numberof individuals in the sample) - Allele frequency = (Number of copies of a particular allele) / (Total number of alleles in the sample) - p (frequency of dominant allele) = frequency of homozygous dominant genotype + (1/2) * frequency of heterozygous genotype - q (frequency of recessiveallele) = frequency of homozygous recessive genotype + (1/2) * frequency of heterozygous genotype - Outline the evolutionary forces and their impact on genetic variation - Mutation - Increases genetic variation - migration/gene flow - Within populations: increases variation - Between populations: reduces variation - Genetic drift - Within: decreases - Between: increase - Examples - Bottleneck - Founder - natural selection - Directional selection - Stabilizing selection - Disruptive selection - Fitness (W) is a measure of a genotype's reproductive success compared to other genotypes in the population, ranging from 0 to 1 - Non-random mating - Can change genotype frequencies but not allele frequencies - Types: - Positive assortative mating - Like individuals mate (more homozygous) - Negative assrtative mating - Unlike individuals mate (more heterozygous) - Inbreeding - Related mates (homozygous and recessive mutations) Quantitative Genetics - Explain the difference between qualitative and quantitative genetics - feature qualitative quantitative traits Discrete, yes/no continuous Gene control one/few genes polygenic Environmental role minimal ignificant influence on S phenotype Phenotypic ranges Distinct, no overlap overlapping - Explain the features of quantitative characteristics - Polygenic - Overlapping phenotype ranges - Environmental influences - Examples: - Meristic characteristics - determined by multiple genetic and environmental factors and can be measured in whole numbers - Threshold characteristics: - traits that are measured by presence or absence - Explain the information that could be gained from a frequency distribution - phenotypes plotted on the x-axis and their frequency on the y-axis - Skewness: - positive skew → few individuals have exceptionally high trait values, - certain alleles contribute to higher trait values, but those alleles are uncommon in the population - negative skew→ few individuals exhibit exceptionally low trait values, - could indicate that certain alleles result in lower trait values, but those alleles remain uncommon - Outliers: - can be indicative of rare genetic mutations or unique environmental factors impacting those individuals - Extremes: - influence of selection or genetic drift within the population - Explain each of the terms in the heritability formula and what it is used for - Heritability: the proportion of the total phenotypic variation that is due to genetic difference - Heritability (broad-sense) H2= VG/VP ranging from0-1 - Can be used to predict how organisms will respond to artificial or natural selection - Determine how environmental changes can impact phenotype - Limitations: - Heritability is specific to a given population in a specific environment - There is no universal heritability for a characteristic - Even when heritbility is high, environmental factors may influence a characteristics - Genetic variance VG=VA+ VD+VI - Additive genetic variance: VA - Additive effects - Dominance genetic variance: VD - non-additive - Genic interaction variance: VI - Interactions between genes at different loci - Phenotypic variance VP=VA+VD+ VI+VE+ VGE - Includes - Environemntal variance: VE - Genetic-environmental interaction: VGE - Explain the general principle of mapping QTL - quantitative trait loci (QTLs) are chromosomal regions containing genes that control polygenic characteristics - The general principle of QTL mapping is to identify associations between genetic markers and phenotypic variation
