Genetic engineering Flashcards
Which are the steps of gene cloning?
- DNA Isolation.
- Produce a specific fragment.
- Clone the specific fragment.
- Self-replicate the fragment/modify.
- Put vector in a host cell.
- Spread cell line.
- Long-term storage.
- Further work of DNA when required.
- Manipulation of DNA fragment into other vectors.
- Investigate gene expression and protein function.
How can cell lysis be achieved?
Use chemicals mixture, detergents, and enzymes to degrade cell walls and membranes.
How can proteins be removed?
Use solvents and salts to remove them from the target DNA.
What happens to the DNA after it is purified?
Concentrated with using alcohol precipitation.
What can careful choices of extraction and purification protocols do?
Target specific types of DNA in complex samples.
How can DNA be visualised?
By gel electrophoresis.
What happens in gel electrophoresis?
DNA is separated into bands based on size, length.
What charge does DNA have?
Negative Electrical.
Where does DNA move?
In an electrical field.
How can DNA be separated?
By using a gel matrix, agarose, acrylamide.
What do shorter molecules do?
Move faster.
Migrate farther.
Migrate easily through pores of the gel.
How can DNA molecules be visualised in the gel?
By using fluorescent dyes.
To what is the distance DNA molecules moved related?
To fragment size.
Where is automated DNA size analysis used?
Diagnostic analyses.
Forensic analyses.
Where is assessing DNA fragment sizes from gels necessary?
In preparatory work.
Research.
How is the size of an unknown DNA fragment determined?
DNA ladder with fragments mixture of known size.
The appropriate chart with the standard curve.
What is used in forensic analyses?
High-quality DNA electrophoresis.
Accurate DNA sizing.
Where is DNA electrophoresis and DNA sizing used in forensics?
Statistically link crime scene DNA samples with victim’s suspects.
Paternity cases.
Medical diagnostics.
Environmental DNA surveys.
What can be very difficult in DNA cloning?
Isolation of DNA piece from rest of the sample.
Where did an understanding of bacterial immunity to bacteriophage infection led?
Discovery of restriction endonucleases. (RE’s)
Who did discover the endonucleases and when?
Arber.
In 1960.
Smith and Nathans.
In 1970.
Where were RE’s used?
Cut DNA in specific lengths –> isolated from gels –> ligated to vector with DNA Ligase.
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