Gene Regulation Flashcards
Liane's Topic L4
What makes cells different?
Cells have different proteins
What is differential gene regulation?
gene regulation through time and space
done by:
-regulate the transcription of gene
-regulate the synthesis and function of a protein post-transcriptionally
How do we know that transcriptional regulation is a commonly used mechanism for differential gene expression during development?
-differential distribution of transcripts within a developing organism (some cells have have certain transcripts)
What are some methods for detecting transcript distribution in an organism?
- RNA (Northern) Blot
- RT PCR
- in situ hybridization
- Microarray hybridization
- RNA sequencing (RNA seq)
- promoter-reporter gene fusion
What is hybridization?
it is a phenomenon in which single-stranded DNA or RNA molecules anneal to complementary DNA or RNA
How does nucleic acid hybridization work?
- A hybridization probe anneals to a single stranded DNA or RNA that is the target
- it can be used to detect the presence of nucleotide sequences (the target)
What is a hybridization probe?
- fragment of DNA or RNA which is radioactively or non-radioactively labeled
- complementary to the target sequence
How do we use RNA (Northern) Blot?
- Isolate RNA from cells from different tissues or the same tissue at different developmental times
- Separate RNA transcript by size using electrophoresis
- Transfer to a hybridization membrane
- Hybridize a labelled gene-specific probe to the RNA on the membrane to detect any RNA molecules that are homologous to the probe
What are the advantages and disadvantages of RNA (Northern) Blot?
Advantage
-provides transcript size and abundance
Disadvantage
-time consuming
What is the RNA dot blot?
- A simplified RNA (northern) blot
- it is to determine if RNA exists
What are the advantages and disadvantages of the RNA dot blot?
Advantage -simple to use -quickly shows if RNA exists Disadvantage -does not tell exactly what type of RNA -does not show the sequence of the RNA
How do you use RT - PCR?
- Isolate RNA from cells from different tissues or the same tissue at different developmental times
- Make cDNA from mRNA using reverse transcriptase and a poly T primer
- Use gene specific primers to amplify cDNA (if the transcript corresponding to the primers is present)
- Run PCR reaction on a gel to observe if there is an amplified product
What are the advantages and disadvantages of using RT-PCR?
Advantage
-can know the exact DNA sequence in relation to the mRNA
-fast, sensitive
Disadvantage
-Need a primer that is minimum 18-24 bp
-Does not work for short DNA
-To use PCR amplification, you need to know the DNA sequence of the nucleic acid to be detected
-No information on transcript size (just know what is ultimately translated)
-Crudely quantitative
-Subject to artifacts (contamination)
What are the steps for in situ hybridization?
- Section organism/organ/tissue of interest
- Hybridize a gene specific probe to a tissue section on a slide
- If any cells within the sectioned tissue have transcripts of the gene of interest (homologous to the probe), the probe will hybridize to those cells. Detection of the prove will identify those cells
What are the advantages and disadvantages of in situ hybridization?
Advantage
-provides precise information on spatial distribution of gene transcript
Disadvantage
-difficult
-time consuming
-little information on amount of transcript
What are the steps of microarray hybridization?
- Synthesize gene specific probes (oligonucleotides) for a large number of genes
- Array the probes on a hybridization membrane or chip
- Isolate RNA from a specific tissue/developmental time
a. Usually a reference sample to see differences in gene expression - Make labelled cDNA from RNA
a. Each sample labelled with a different fluorescent dye - Hybridize the labelled cDNA to the array of probes and detect which probes hybridize to the population of cDNAs
What are the advantages and disadvantages of microarray hybridization?
Advantage
-provides info on the amount of RNA transcript for every gene included in the array
Disadvantage
-expensive
-results must be repeated or verified by another technique
Why is RT-PCR not very quantitative?
The amount of DNA product reaches a plateau that is not directly correlated with the amount of target DNA in the initial PCR
How do we resolve the issue with RT-PCR not being very quantitative?
- include a probe with a reporter that fluoresces only when new DNA is synthesized.
- The amount of fluorescence measured shows the total amount of amplified DNA present
- Analyze how fluorescence changes with PCR cycle
What are the steps of RNA sequencing?
- Isolate RNA from cells from different tissues or the same tissue at different developmental times
- Make cDNA from mRNA using reverse transcriptase
- Sequence the cDNA population (with Next Gen Methods)
What are the advantages and disadvantages of RNA sequencing?
Advantage -sensitive -quantitative -accurate -provides information on all transcripts Disadvantage -relatively expensive
What is an example of next generation sequencing?
illumina genome analyzer
What are the advantages of using next generation sequencing?
- Not based on electrophoresis
- Millions to billions of sequence reactions in parallel (massive parallel sequencing)
- Sequences are generally short
- Cost per base pair is very lower
- Many potential applications
What are the steps of next generation sequencing?
- DNA is fragmented
- Adaptors ligated to fragments
- Generate array of PCR colonies by Bridge PCR
- Sequence
a. Enzymatic extension with fluorescently tagged nucleotides
b. The identity of each base of a cluster is read off from sequential images of the array
What is bridge PCR?
- PCR carried out on fixed surface
- generate array of PCR colonies
- used in next generation sequencing
What are the steps of Bridge PCR?
- prepare genomic DNA sample. Randomly fragment genomic DNA and ligate adapters to both ends of the fragments
- attach DNA to surface. Bind single stranded fragments randomly to the inside surface of the flow cell channels
- fragments become double stranded
- denature the double stranded molecules
- completion of amplification. On completion, several million dense clusters of double stranded DNA are generated in each channel or the flow cell.
What are some key ideas of bridge PCR?
- randomly generated DNA fragments are ligated to adaptors
- one end of each DNA fragment is fixed to a solid surface
- this surface is also coated with forward and reverse PCR primers that correspond to the adaptors
- amplification proceeds in cycles, with one end of the DNA tethered to the surface
- after several cycles, each amplified genomic fragment results in a cluster of fragments on the surface
What is sequencing? (In next gen?)
enzymatic extension with fluorescently tagged nucleotides. The identity of each base of a cluster is read off from sequential images of the array
What are the steps in sequencing by synthesis?
- Cycle 1
2. Cycle 2-n
What happens in cycle 1 of sequencing by synthesis?
- add sequencing reagents
- first base incorporated
- remove unicorporated bases
- detect signal
- cleave block and fluorescent groups (this is done by reversible terminator chemistry)
What happens in cycle 2-n?
adding sequencing reagents and repeat
What are the steps of promoter-reporter gene fusion?
- Construct a chimeric gene using a reporter gene under the control of a promoter and regulatory sequences from a gene of interest
- Transform the chimeric gene into the organism of interest such that all cells carry the chimeric (transgenic) gene
- Detect which cells are expressing the reporter gene
What are the advantages and disadvantages of promoter-reporter gene fusion?
Advantage
-determines in which cells the gene of interest is transcribed in
Disadvantages
-time consuming, must know which sequences contain all the promoter regulatory sequences (enhancer)
What are vectors for cloning?
a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed
How do you insert a reporter gene?
- remove the gene (exons and introns, everything after the promoter)
- insert the reporter gene after the promoter
usually the reporter gene is lac Z
What technique used for analyzing transcript abundance in global gene expression analysis?
- microarray
- RNA sequencing
What technique is used for analyzing transcripts in analysis of expression of individual genes?
- RNA blot
- RT-PCR/Real-time RT-PCR
- In situ hybridization
- Promoter-reporter gene fusion
How can transcription be used to control transcript levels?
- The promoter and enhancer regions of genes with specific patterns of transcript localization can be used to express reporter genes in the same pattern
- If the promoter and enhancer regions of a gene (x) can be used to transcribe a reporter gene (Y) in the same tissue specific pattern as X, then the differential transcript pattern should result from the regulation of transcription
How do we know that transcription regulation is a commonly used mechanism for differential gene expression during development?
- differential distribution of transcripts within a developing organism
- transcription (not RNA degradation) is the principle mechanisms for controlling transcript levels
- transcription factors are abundant and many are required for development
What are the types of transcription factors?
- general transcription factors: bind to a core promoter and required for transcription initiation
- specific transcription factors: bind to other sequencings (enhancer, repressors) to regulate transcription of specific genes
What is a transcription factor?
a protein that influences RNA polymerase to increase or decrease transcription of a gene
How do we know transcription factors are abundant and many are required for development?
- sequencing of entire genomes has shown that 10-20% of all genes in a genome encode transcription factors
- mutants disrupted in development identify genes that play key regulatory roles during development
What kinds of evidence do you need to prove that gene y is a target gene of transcription factor x?
- mutating X affects the expression of Y
- X can bind to cis elements in the regulatory sequence of Y in vitro
- x binds to the regulatory sequence of Y in vivo
What is a DNA probe?
- something that is labelled
- contains the TF recognition site
- TF-bound probe is larger than free probe so it doesn’t travel through a gel as far
What are the steps to ChIP -PCR?
- Take cross link cells and cause cell lysis
a. Proteins and DNA are chemically linked to preserve interactions - Sonication
a. Hit the DNA with the high frequency sound waves used to fragment DNA - Immunoprecipitation
a. Antibodies (bound to beads) are used to identify and remove protein of interest (and bound DNA) - Bead-antibody-protein-target DNA complex
- Protein Digestion and DNA purification
- qPCR ChIP - Seq
• qPCR on isolated DNA to quantify amount of DNA originally bound by TF of interest
• DNA sequencing to identify sequence/element that is bound by TF of interest
