Why is noncoding RNA also called functional RNA?
noncoding RNA molecules do not contain instructions for protein synthesis. However, they are involved in regulating gene expression and
Gene regulation
RNA Processing and Modification
Structural and Catalytic Functions
Epigenetic Regulation
How can proteins have so many diverse structures and functions when they are all synthesized from just 20 amino acids?
amino acid sequence
(protein folding)
(Post-Translational Modifications)
also Amino acid side chains are diverse, with different properties that determine their functions
What are two important chemical differences between RNA and DNA?
RNA contains ribonucleic acid while DNA contains deoxyribonucleic acid (DNA has fewer oxygen atoms per nucleotide)
RNA has the nucleobase uracil while DNA contains thymine
(also DNA is double-stranded while most RNA is single stranded)
In addition to the 20 amino acids, proteins have additional chemical diversity because of two factors. What are these 2 factors and what is their importance?
Post-Translational Modifications
protein folding
How do primers determine the specificity of PCR?
The primers define the flanking regions of the target so if those flanking regions closer to the target. then the PCR is more specific.
Describe how a DNA fragment containing a single gene of interest would be obtained by
(A) PCR or
(B) gene cloning?
(A) design primers, obtain sample with target gene, do PCR amplification
(B) isolate DNA containing target, use restriction enzymes to cut DNA,Prepare a plasmid vector that will be used to clone the gene, ligate vector with target gene, Transformation, Select the transformed cells using antibiotic selection
Why are vectors that can carry larger DNA inserts beneficial for the creation of clone libraries?
capture of full-length genes, reducing fragmentation, providing better representation of complex genomes, enabling functional analysis of genomic regions, simplifying cloning procedures, and supporting comprehensive studies of gene regulation and functional genomics.
Explain why bacterial plasmids are popular cloning vectors.
they are small and easy to manipulate
can be easily introduced into bacterial cells
Plasmids often replicate autonomously within bacterial cells, resulting in a high copy number of plasmids per cell
typically carry selectable markers, such as antibiotic resistance genes
Stability and Inheritance
Explain why SNPs are now the most widely used type of DNA marker, and outline the various methods used to type SNPs.
abundance, high variability, and widespread distribution throughout the genome
SNP typing methods offer flexibility, scalability, and high-throughput capabilities
Methods:
allele-specific PCR
RFLP analysis
DNA microarrays
NGS technologies
How has PCR made the analysis of RFLPs (Restriction fragment length polymorphism) much faster and easier?
we can now selectively amplify specific DNA fragments, eliminating the need for large amounts of starting material
PCR eliminates the need for laborious and time-consuming cloning steps, enabling direct analysis of the amplified DNA fragments
How would a scientist prepare a clone library of DNA from just a single chromosome?
Isolation of the chromosome
DNA extraction
Fragmentation of DNA
Construction of a vector
Ligation and transformation
Selection and screening
How has DNA “chip” technology aided biological research.
DNA “chip” technology enabled high-throughput analysis of gene expression, genotyping, and DNA-protein interactions
allows researchers to simultaneously analyze the expression levels of thousands of genes or detect variations in DNA sequences in a single experiment
Explain how the inclusion of dideoxynucleotides in strand-synthesis reaction enables a DNA sequence to be read.
By including all four types of ddNTPs (ddATP, ddCTP, ddGTP, and ddTTP) in the reaction mixture, along with the regular dNTPs, the DNA synthesis process will sometimes be terminated at every position along the DNA strand, resulting in a collection of DNA fragments of different lengths.
These fragments are then separated by size using gel electrophoresis, where shorter fragments migrate faster through the gel than longer ones. The DNA fragments can be visualized using fluorescent dyes or radioactive labels. The order of the fragments on the gel corresponds to the sequence of the original DNA template.
Compare the strengths and weaknesses of chain-termination and next-generation sequencing methods.
Chain-termination (Sanger)
strengths: high accuracy, low error rate
weaknesses: limited throughput, less suitable for large-scale sequencing projects
NGS:
strengths: high throughput, allowing for the simultaneous sequencing of millions of DNA fragments, greater sequencing depth
weaknesses: higher error rates
How is a sequencing library prepared during a next-generation sequencing project?
DNA fragmentation
Adapter ligation
PCR amplification
Library quality control
What factors complicate the application of shotgun sequencing to a eukaryotic genome?
Genome size, Repetitive regions, Alternative splicing and complex gene structures
Describe how sequence reads are assembled into contiguous reads.
Read preprocessing
Overlap detection
Graph construction (de Bruijn)
finding eulerian path
Define the term “ homologous” when comparing genome sequences.
sequences or genes that share a common ancestral origin
exhibit similarity or conservation of sequence across different organisms or within the same organism
(orthologs vs paralogs)
Describe a method for the experimental identification of exon-intron boundaries.
compare results of DNA-seq and mRNA-seq
What are the 4 current methods being used for whole genome sequencing?
Sequencing by synthesis (Illumina)
Pyrosequencing
Long Read sequencing nanopore
Long Read sequencing PacBio
How are different RNA’s targeted sequenced?
there is:
mRNA seq
Small RNA seq
Ribo-seq
total RNA-seq
you can target a specific RNA by poly-a-tail selection(mRNA),
ribo-depletion (everything but rRNA), size selection (small RNA)
How is the CRISPR system used to inactivate eukaryotic genes?
CAS complex including a guideRNA and a PAM is introduced into cell/organism
Cas9 protein acts as a molecular scissors, while the gRNA provides the sequence specificity to guide Cas9 to the target gene
this way a gene can be knocked out very easily
What techniques would be used to locate and sequence DNA bound to a protein?
ChIP-Seq
(because it is antibody specific we also use ELISA here)
specifically ribosomes: riboseq
What does the treatment of eukaryotic chromatin with nucleases reveal about the packaging of eukaryotic DNA?
It reveals regions of open chromatin and accessibility, nucleosome positioning, and the compaction status of chromatin, shedding light on the regulatory mechanisms and functional elements within the genome.
Explain why it is important that chromosomes have telomeres at their ends.
Chromosome Stability
DNA End Protection
What types of repetitive DNA are present in the human genome?
Transposable Elements (LINE, SINE: long/short interspersed nuclear elements)
Satellite DNA
Minisatellites and Microsatellites
Explain how specific RNAs are targeted for degradation by siRNA and miRNAs.
siRNAs, introduced exogenously, guide the RNA-induced silencing complex (RISC) to cleave the target RNA, while endogenous miRNAs guide the RISC complex to partially complementary target mRNAs, resulting in translational repression and mRNA degradation
What is alternative splicing and how is it regulated?
exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcripts
regulation:
trans-acting proteins (repressors and activators) and corresponding cis-acting regulatory sites (silencers and enhancers) on the pre-mRNA
Describe how components of a proteome are separated prior to the mass spectrometry stage of protein profiling.
Gel Electrophoresis: separates proteins based on their isoelectric point and size
Liquid Chromatography: separates proteins based on their chemical properties, such as size, charge, hydrophobicity, or affinity
What methods are available for identification of proteins that interact with one another?
co-immunoprecipitation (co-IP), pull-down assays, crosslinking, label transfer, and far–western blot analysis
Your PI comes to you and asks your advice on techniques for sequencing a novel prokaryotic genome. Please support your idea with scientific reasoning.
Use Illumina Sequencing because it has very high read accuracy and for a novel genome we want high accuracy
Your PI comes to you and asks your advice on how to determine the function of a novel gene.
Please support your idea with scientific reasoning.
Gene Expression Analysis (qPCR): can determine when and where the gene is expressed
Gene Knockdown: see what happens when gene is knocked out
Your PI comes to you and asks your advice on how to determine the function of a novel protein.
Immunocytochemistry/Immunohistochemistry: see where protein is expressed
check Protein-Protein Interactions with co-immunoprecipitation
Give 2 examples of how scientists have used homologous recombination for the benefit of research.
Gene Targeting: introduce precise modifications in the genome of model organisms such as mice (designing a targeting vector with homology to a specific gene of interest)
Protein Engineering: Protein engineering with homologous recombination develops chimeric proteins by swapping fragments between two parental proteins
Your PI comes to you and asks your advice on how to test if separate genes are interacting together. Please support your idea with scientific reasoning.
do Hi-C because it tells you if the genes are in close proximity in 3D which could be an indicator that they are interacting with each other
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