Molecular Genetics exam 4

Evaluate RNA samples for “fit for purpose” use in downstream applications considering integrity, purity, and potential biases

→ RT-PCR: Moderate Quality OK

→qPCR: High purity + no DNA

→RNA-seq: High integrity (RIN > 7)

→Cloning cDNA: Full length transcripts

Explain how structural features of eukaryotic mRNA (5’ cap and poly-A tail) enable selective purification of mRNA from total RNA

Eukaryotic RNA is unique because of its 5’ cap and 3’ poly-A-tail

These features can be selected for out of all of the other RNA through affinity chromatography or magnetic bead based separation

Compare strategies for mRNA enrichment, including poly(A)-based selection, and justify their use in different experimental context

poly(A)-based selection: Oligo(dT) beads hybridize to poly(A) tails. It targets mature, polyadenylated eukaryotic mRNA and requires high quality RNA

rRNA depletion: Probes hybridize to rRNA, followed by removal, Targets poly(A) and non-poly(A) RNAS such as non-coding RNA and immature mRNA

Describe the molecular mechanism of cDNA synthesis, including first-strand and second-strand synthesis and the roles of reverse transcriptase and DNA polymerase

Core components:

1. Template RNA

2. Primer

3. Reverse transcriptase

4. dNTPS

5. Buffer

First strand: Reverse transcriptase (an RNA-dependent DNA polymerase) reads the RNA template and synthesizes a DNA strands

Second strand: RNase H removes the original RNA template, and DNA polymerase synthesizes the complementary DNA strand to create a double helix

Design an appropriate cDNA synthesis strategy by selecting primer types based on RNA quality and experimental goals

Oligo(dT) primers: Use if you want only mRNA and full-length transcripts

Random Primers: Use if the RNA is degraded of if you want to sequence all RNA types

Gene-specific: Use for RT-qPCR when you only care about on specific target gene

Identify sources of bias and error in cDNA synthesis and predict their impact on downstream results

1. RNA degraded

2. Wrong primer choice

3. DNA contamination

4. Poor enzyme activity

Explain how DNA microarrays are constructed from genomic or cDNA sequences

1. Select genes: an entire genome or a few unique genes that represent selected species

2. Generate DAN probes: cDNA or synthesized oligos

3. Spot on slide: robotic printing onto glass chip. Each spot = one oligo = one gene

4. Organized grid: Known position = known gene identity

General workflow

1. Do the experiment: treat cells with control and experimental conditions

2. RNA isolation: from both control and experimental conditions

3. Prepare RNA for hybridization to DNA microarray: reverse transcription. Fluorescent labeling

4. DNA microarray (aka=chip: incubate labeled cDNAs from samples with chip. Wash to remove non-hybridized materials

5. Scanner

Describe how labeled cDNA hybridized to probes to measure gene expression

1. Isolate mRNA

2. Covert to cDNA

3. Label cDNA with fluorescent dyes: Conditions A is green while condition B is red

4. Hybridize to chip

5. Wash and scan fluorescence

Interpret microarray output data (heat maps, fold change)

→ Two dimensional, color-coded grid visualizations

→Transforms thousands of gene expression values into format

→Map expression levels to color: red is high, green/blue for low and black for no detected mRNA

→Uses: identify clusters of co-regulated genes. Compare experimental samples. Detects, upward or downward, shifts in expression patterns

Design a basic microarray experiment with appropriate controls

:D

Key considerations:

1. Biological question: ex stress response in yeast

2. Controls: replicates (biological and technical)

3. Normalization: correct for technical variation

4. Statistical analysis: avoid false positives

Evaluate strengths and limitations of microarrays vs RNA-seq

Limitations of microarrays

1. Requires known sequences

2. Limited dynamic Range

3. cross-hybridization (noise)

4. cannot detect novel transcripts

5. Quantitative accuracy < RNA-seq

Applications of microarryas

1. Disease profiling (cancer subtypes)

2. Drug response

3. Environmental stress response

4. functional genomics

5. infectious disease diagnosis

Microarrays vs RNA-seq

1. Microarray: No novel genes, moderate sensitivity and lower cost

2. RNA-seq: Yes novel genes, high sensitivity and higher cost

Use microarray data from SGD to generate hypotheses about gene function

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Define sense and antisense RNA

Antisense: An RNA molecule that is complementary to the mRNA

Sense RNA: Normal RNA that has been produced from the noncoding strand of DNA

describe how bacterioferritin synthesis regulated by antisense RNA

Bacterioferritin: Bfr, encoded by bfr. It regulates the storage and utilization of iron, which is essential for the growth and metabolism of organisms

When iron is plentiful: antibfr gene is not expression and only the bfr mRNA is produced, Bfr protein is made

When iron is low: both bfr and antibfr mRNA are expressed. RNAi and no Bfr protein is made

RNA interference

→Triggered by the presence of dsRNA and results in the degradation of mRNA or other RNA transcripts homologous to inducing dsRNA. It is conserved across eukaryotes

Mechanism: RNAi is a naturally occurring cellular process where dsRNA is converted into small interfering RNAs (siRNAs), which then guide cell machinery to degrade complementary mRNA transcripts and suppresses gene expression

1. dsRNA is introduced

2. Dicer processes into siRNAs

3. siRNA loads into RISC

4. RISC targets and cleaves mRNA

Describe the function of Dicer, RISC, siRNA and Argonaut

Dicer: Cleaves dsRNA into siRNAS. It is an enzyme from the RNase III family. It processes dsRNA or pre-miRNA into 21-23 nucleotide siRNA or miRNA

RISC: “RNA-induced silencing complex”. It mediates gene silencing

siRNA: “short interfering RNA”. Guides RISC to target mRNA

Argonaut: core slicer protein in RISC. Associate with small non-coding RNAs such as siRNA and miRNAs. Function in RNA-based silencing mechnaisms by altering protein synthesis and affecting RNA stability. It can even participate in the production of a new class of small non-coding RNAs known as piwi-interacting (pi)RNAs. Its a core component of RISC and has slicer activity for target mRNA cleavage. It contains four conserved domains: the N terminal, PAZ (Which is a responsible for small RNA binding), Mid and PIWI (which confers catalytic activities) domains

Describe microRNA and its effect on gene expression

microRNA: Endogenous small RNA transcribed as hairpin precursors. Regulates gene expression by imperfect pairing of mRNA. Binds 3’ UTR of target mRNAs and causes translation repression or mRNA degradation

1. Transcription →pri-miRNA

2. Drosha processes→ pre-miRNA

3. export to cytoplasm

4. dicer cleaver → mature miRNA

5. load into RISC

Differentiate between siRNA and miRNA

siRNA: exogenous, perfect match, cleavage

miRNA: endogenous, imperfect match, repression

Both processed by Dicer and function via RISC

Describe how RNAi can be used experimentally to prevent gene expression

RNA interference (RNAi) = post-transcripional gene silencing

Uses small RNA molecules: siRNA that targets specific mRNA for degradation that results in reduced or no protein expression

→dsRNA introduced into a cell, processed by dicer→ siRNA. siRNA loaded into RISC complex. Guide strands bind complementary mRNA and specific mRNA is cleaved and degraded.

1. determine gene function

2. study essential genes

3. validate targets from microarray/RNA-seq data

4. pathway anlysis

Real world

1. Disease research

2. Antiviral straegies

3. Functional genomics screens

Explain the purpose of RNA-seq

Its a method to sequence and quantify RNA molecules within a sample. It provides a snapshot of the transcriptome.

It answers

1. Which genes are expressed?

2. How much are they expressed?

3. Are there alternative transcripts?

Outline the workflow for RNA-seq, scRNA-seq and Drop-seq

RNA-seq:

1. RNA isolation

2. mRNA enrichment

3. cDNA synthesis

4. Library preparation

5. sequencing

6. data analysis

ScRNA-seq

1. Isolate individiual cells

2. capture RNA from each cells

3. convert to cDNA

4. add cell-specific barcodes

5. pool and sequence

6. assign reads back to original cells

Drop-seq

-1. AFter obtaining sequencing reads consisting of cell barcode, UMi and cDNA

2. first group reads by cell abrcode before aligning cDNA reads

3. Counting unique molecules per cell per gene using UMIs

4. Estimate the transcript abundances

Distinguish RNA-seq from older methods (ex. microarrays)

RT-PCR: its low throughput

Microarrays: require known sequences

RNA-seq: unbiased and genome wide

Interpret basic RNA-seq outputs

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Describe key applications of RNA seq in research and medicine

1. Disease mechanism discovery

2. biomarker identification

3. cancer diagnostics

4. cell development

5. cell-specific responses

Describe how the CRISPR system protects bacteria from viruses

Captures, stores and utilizes snippets of viral DNA to identify and destroy future infections

→Adaption: acquisition of new sequences

→Expression: stored sequences expressed during a subsequent infection

→Interference: effector complex targets and cleaves incoming foreign DNA

Describe how CRISPR systems are classified

Class One Systems: Common in nature and more complex

Class Two systems: rare in nature and less complex. Requires just one Cas protein, Cas9 nuclease, and two RNA components (tracrRNA and crRNA)

Differentiate between PAM and PFS

Both are short flanking DNA or RNA sequences required for CRISPR-case systems to identify, bind and cleave targets

PAM: Protospacer adjacent motif: used for DNA targeting systems like Cas9. It is required for Cas nucleases to distinguish between bacterial CRISPR arrays(no PAM) and viral DNA (contains PAM). Double stranded DNA

PFS: associated with RNA-targeting systems. Often regulate cleavage activity. single-stranded RNA

Describe an sgRNA and how it is created

→help to target specific DNA sequence in a gene of interest

→must be adjacent to PAM sequence

→Minimize off-target matches

Define the different components necessary for gene editing by CRISPR

→Cas9 Protein (DNA-cutting enzyme)

→guide RNA (gRNA) sequence-specific targeting

For CRISPR, define the term off-targets

unwanted effects from CRISPR targeting

Describe the basic steps of a CRISPR experiment

1. Define your goal

2. Design the Guide RNA (gRNA)

3. Choose CRISPR System

4. Deliver components into cells

5. Screen and validate results

Describe how CRISPR can be used experimentally to alter gene expression

Transcriptome

set of all RNA moelcules, including mRNA, rRNA, tRNA and other non-coding RNA< produced in one or a population of cells

Proteome

Total set of proteins encoded by a genome or the total protein complement of an organism

Translatome

total set of protein that have actually been translated and are present in a cell under any particular set of conditions

Isoelectric focusing

high-resolution electrophoretic technique that separates proteins or peptides in a gel based on their unique isoelectric point (PI)

Provide reasons for the discrepancy between: proteome and translatome, proteome and transcriptome

Transcriptome and proteome:

1. Some RNA molecules are non-coding

2. Alternative splicing →multiple protein products

3. Levels of mRNA may not correlate with protein levels due to differential rates of mRNA translation or degradation

4. Regulation of proteins by additions of PTM

5. Regulation of protein by chemical modifications

6. Modifications by proteolytic cleavage or addition of sugar or lipid residues to give glycoproteins or lipoproteins

7. Proteins themselves may be degraded and vary greatly in stability

Proteosome and translatome

1. Promteosome: entire set of proteins expressed by genome cell, tissue or organisms

2. Translatome: subset of mRNAs that are actively being translated into proteins by ribosomes

Identify the essential components of an expression vector used for eukaryotic expression systems

1. Strong promoter

2. Ribosome binding site

3. selectable marker

4. tag sequence

5. PolyA site

Describe the basic steps in recombinant protein expression

1. Clone gene into expression vector

2. Introduce into host cell

3. Induce expression

4. Harvest cells

5. Isolate protein

Compare pros/cons of bacterial/insect/mammalian expression systems

Bacteria:

→Pros: Fast growth rates, inexpensive, easy to scale up for high volume production, well-understood genetics

→Cons: lack machinery for complex PTMs, frequent formation of insoluble inclusion bodies, often requires difficult refolding steps

Insect:

→Pros: capable of complex PTMS, higher yield than mammalian cells, safter to use than mammalian pathogens, efficient of multi-protein complexes

→Cons: time-consuming to develop the virus, more expensive than bacteria

Mammalian

→Pros: provides native human-like PTMs and glocosylation, proper protein folding, high bioactivity, and secretion into media for easier purification

→Cons: very expensive, slow growth, lower yields, fragile cell lines require strict culturing conditions

Compare/contrast transient and stable transfections

Transient: DNA is not integrated into the genome but remains within the nucleus. Genetic material is not passed onto progeny and genetic alternation is not permanent

Stable transfection: DNA integrates into the genome. Transfected material is carried stably from generation to generation; genetic alteration si permanent

Define transfection and transduction relative to mammalian expression vectors

Transfection: Non-viral. Uses agents or physical forces to force DNA across the cell membrane

Transduction: foreign DNA is introduced into a cell via a viral vector

Design a basic mammalian expression strategy

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Describe the following inducible promoter systems: lacUV, T7, Tet-on, Tet-off

lac UV:

1. Components: Lac promoter, LacI repressor, Isopropyl B-d-1 thiogalactopyranoside (IPTG) as an inducer

2. Default stage is off and when inducer is added turns on

T7:

1. T7 promoter, T7 RNA polymerase, very strong transcription

2. Very useful when you want high protein production in bacteria

Tet-ON

→Tet added→ transcription ON

Tet-OFF

→Tetracycline added→ Transcripion off

Differentiate types of chromatography discussed

For each of the protein tags: describe their structure and how they work in protein purification

Differentiate: polyclonal and monoclonal antibodies, primary and secondary antibodies

Differentiate between native and denatured proteins

Describe the functions of the following: SDS-PAGE, SDS, B-mercaptoethanol, Coomassie blue, ponceau S

describe western blotting

compare and contrast methods for localizing a specific protein in cells