Old questions

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

amino acid sequence

(protein folding)

(Post-Translational Modifications)

also Amino acid side chains are diverse, with different properties that determine their functions

1. RNA contains ribonucleic acid while DNA contains deoxyribonucleic acid (DNA has fewer oxygen atoms per nucleotide)

2. RNA has the nucleobase uracil while DNA contains thymine

(also DNA is double-stranded while most RNA is single stranded)

Post-Translational Modifications

protein folding

The primers define the flanking regions of the target so if those flanking regions closer to the target. then the PCR is more specific.

(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

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.

• 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

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

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

1. Isolation of the chromosome

2. DNA extraction

3. Fragmentation of DNA

4. Construction of a vector

5. Ligation and transformation

6. Selection and screening

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

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.

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

1. DNA fragmentation

2. Adapter ligation

3. PCR amplification

4. Library quality control

Genome size, Repetitive regions, Alternative splicing and complex gene structures

1. Read preprocessing

2. Overlap detection

3. Graph construction (de Bruijn)

4. finding eulerian path

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)

compare results of DNA-seq and mRNA-seq

Sequencing by synthesis (Illumina)

Pyrosequencing

Long Read sequencing nanopore

Long Read sequencing PacBio

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)

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

ChIP-Seq

(because it is antibody specific we also use ELISA here)

specifically ribosomes: riboseq

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.

1. Chromosome Stability

2. DNA End Protection

Transposable Elements (LINE, SINE: long/short interspersed nuclear elements)

Satellite DNA

Minisatellites and Microsatellites

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

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

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

co-immunoprecipitation (co-IP), pull-down assays, crosslinking, label transfer, and far–western blot analysis

Use Illumina Sequencing because it has very high read accuracy and for a novel genome we want high accuracy

Gene Expression Analysis (qPCR): can determine when and where the gene is expressed

Gene Knockdown: see what happens when gene is knocked out

Immunocytochemistry/Immunohistochemistry: see where protein is expressed

check Protein-Protein Interactions with co-immunoprecipitation

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

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