Applications of Genetic Engineering Flashcards
What are the two general steps in genetic engineering?
1) Target identification
2) Target validation
What are the two examples of target identification?
- Microarray
- Yeast-2-hybrid screen
What are the two examples of target validation?
- Gene knockdown using siRNA
- Gene knockout in transgenic mice
If some neurones became hypersensitive when damaged how would you find out why?
You would compare damaged and undamaged neurones
Why are microarrays not always possible?
Need whole genome sequenced
What is a microarray?
An in vitro (cell-free) technique which allow you to monitor the expression of 1000s of genes at one. Uses isolated mRNA
How many probe cells does the gene chip contain?
45,000
What must be known for a microarray to work?
The location of each probe for a corresponding gene is known
Which gene is being used
What is the procedure of a microarray?
- Extract mRNA from a control and experimental sample
- mRNA –> cDNA with the control labelled with fluorescent green dye and experimental sample labelled with a fluorescent red dye
- Labelled cDNAs mixed and allowed to hybridise with the microarray’s complementary probes
- DNA sequence represented by each spot is different and tract by the computer
- After incubation, the microarray is washed and scanned
What does a grey dot portray on a microarray?
Little or no expression
What does a yellow dot portray on a microarray?
Equal amounts of control and experimental sample hybridised/expressed
If the experimental sample gene was expressed at a much higher level than the control sample, what colour would the dot be?
Red
What enzyme is associated with the process of converting mRNA to cDNA?
Reverse transcriptase
Which technique would you use to find out what protein regulates a certain protein of interest?
Yeast-2-hybrid screen
Process of yeast-2-hybrid screen
- Start with a known protein of interest (POI)
- POI is bound to binding domain on promoter region
- Proteins that could potentially bind to the POI are bound to an activator-binding domain
- Using a library of cDNA with fusions between unknown proteins and activator-binding domain, mix.
- When two proteins bind, the activator0bindong domain and DNA-binding domain are in close proximity —> transcription
- Then you can grow in cultures to see if they survive by expressing the genes they need in order to survive
- Then isolate surviving colonies and isolate plasmic contains edna for sequencing
