Levels Of Gene Control Flashcards
What is the result of differential gene expression
- Results in when, where and how much of a particular transcript and/ or protein is formed.
- Results in the differentiation of organs and tissues in embryonic development.
What causes differential gene expression?
Differential gene expression is a response to paticular cellular signals (growth factors, hormones) as well as environmental changes (available nutrients, exposure to heat).
Gene expression is controlled at different levels.
- Genome level
- Transcription level
- Post-transcription level
- Translation level
- Post-translation level
Gene expression at Genome level
-number of gene copies available
Gene expression at Transcriptional level
Number of mRNA copies transcribed per gene.
Gene expression at Translation level
Number proteins translated per mRNA copy.
Gene expression at Post-translational level
Number of protein copies modified after translation.
How to investigate and demonstrate gene control.
- You can study regulation on mRNA level.
- You can study control on protein level
- You can study control on DNA level.
Techniques to study regulation on mRNA level.
- Northern blotting (an individual transcript)
- QRT-PCR
- Microarrays (populations of mRNA )
Techniques to study control on protein level
- PAGE
- Western blotting (individual protein)
- 2D PAGE(total protein composition)
Techniques to study control on DNA level
- Southern blotting
2. DNA sequencing
Methods to investigate protein expression.(western blotting)
Step 1: separate complex mixture of proteins within cells using gel electrophoresis and stain proteins.
Step 2: “ Probe” for a particular protein using antibodies.
How to “Probe” for a particular protein using antibodies.
- Transfer the separated proteins to a synthetic membrane.
- Incubate membrane with an antibody to that specific protein.
- Detect using autoradiography (X-ray)
What are the steps of two dimensional polyacrylamide electrophoresis.
- Separation of proteins by pl value.
- Soaking the gel in SDS solution and fitting it on an SDS PA gel.
- Separating the proteins by molecular mass with SDS PAGE .
What are the steps tandem mass spectrometry
- Remove protein spot from 2D gel.
- Digest proteins to peptides
- Analyse by mass spectrometry. It converts peptides into ions and then measure their molecular weights
- Use these weights to search database of known proteins, identifying the protein producing the spot.
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Process of Reverse transcriptase PCR (RT-PCR) to detect transcription of a particular mRNA.
- MRNA is converted into cDNA copy by enzyme reverse transcriptase.
- Gene specific primers then used in PCR to amplify that specific cDNA.
- If mRNA of interest is present, it will be amplified and can be visualised as band of an expected size on an agrose gel.
Quantitative RT-PCR
Fluorescent dye (e.g. SYBR green) is added to PCR step, special thermocycler measures exact quantities at every cycle using a lazer a detector.
Advantages of RT-qPCR
- Can detect very low levels of transcript
- Is highly sensitive, gives very accurate quantification.
- Can compare levels of either one gene-specific transcript, or levels of a few different transcripts, between samples.
Steps of microarray
- MRNA from diseased tissue and normal tissue are converted into cDNA and the then respectively fluorescently labeled.
- Microarray contain many know DNA sequences in certain blocks on the glass slide. The CDNA of are diseased and normal tissue are passed along the microarray. If they are a match to the DNA on the microarray the will bind if not they will be removed from the glass slide.
- Scanner will scence the fluorescent signals of the cDNA . IF ONLY HEALTHY cDNA = GREAN if only diseased cDNA= red, if both = yellow.
