Manipulation of Genes and Cells Flashcards
What enzyme is used to isolate and purify a gene from a large DNA molecule?
Restriction nucleases
What reaction does a nuclease catalyse?
Hydrolysis of a phosphodiester bond in a nucleic acid.
What is a special property of restriction nucleases?
They cut the double-stranded DNA only at particular sites.
What is the importance of these restriction nucleases?
They can be used to produce a reproducible set of specific DNA fragments from any genome
Where are these nucleases obtained from?
They are obtained from bacteria.
How can DNA fragments be separated from one another?
Gel electrophoresis
How are DNA fragments separated in electrophoresis?
By their length
How does electrophoresis work?
A voltage is applied across the gel. The DNA molecule is negatively charged and the larger fragments will migrate more slowly because their progress is impeded.
How is DNA sequenced?
Dideoxy method
What happens in the dideoxy method?
DNA polymerase is used to make partial copies of the DNA fragment to be sequenced. This replication reactions are performed under conditions to ensure that the new DNA strands terminate when a given nucleotide (A, G , C or T) are reached. This method produces a collection of different DNA copies that terminate at every position in the original DNA and differ in length by a single nucleotide. These can then be separated using electrophoresis and ordered.
What is nucleic acid hybridisation?
A technique in which single-stranded nucleic acids (DNA or RNA) are allowed to interact so that complexes called hybrids are formed by molecules with similar or complementary sequences.
Why is the high specificity of nucleic acid hybridisation important?
We can determine the exact nucleotide change e.g. in sickle cell anaemia. Makes scanning through the human genome a lot more simple.
What is cloning of DNA?
It is the act of making identical copies of a DNA molecule.
What is transformation?
Introduce DNA to be copied into rapidly dividing bacterium. For example E. coli can be coaxed to take up recombinant DNA made up in the laboratory.
What happens when the modified DNA is inside the recipient cell?
The donor DNA can become a part of the recipient genome (e.g. through homologous recombination) or it can be maintained as a piece of DNA independent to that of the bacterial chromosome.
Why are plasmids useful?
Because it is easier to manipulate, copy and purify recombinant DNA when it is maintained with certainty as an independent molecule., separate from the bacterial chromosome.
What are useful properties of a plasmid?
- it has a cutting site for a convenient restriction nuclease so that the plasmid can be opened and a foreign DNA fragment can be inserted.
- They contain some selectable property such as antibiotic resistance which means that the bacteria that take up the recombinant DNA are easily identified.
How can the DNA fragment be recovered from the plasmid DNA?
By being cleanly cut out using a restriction enzyme and then separated from plasmid DNA by gel electrophoresis.
How are human genomic libraries constructed?
Using restriction nucleases and ligase.
What compromises of a human genomic library?
A set of bacteria, each carrying a different small fragment of human DNA
What is different about the cDNA library?
The complementary DNA library does not contain any introns.
How is the cDNA library created?
total mRNA is extracted from cells and a DNA copy is made using reverse transcriptase. DNA polymerase is then used to make a complementary DNA strand. mRNA does not contain any intron information.
What is PCR used for?
The amplification of selected DNA sequences to make multiple copies. Special heat resistant DNA polymerase is used so it will not denature as DNA strands are heated after each cycle.
Describe DNA engineering
Splice together DNA fragments derived from different sources. After each DNA insertion step, recombinant plasmid is cloned to purify and amplify new DNA. Recombinant molecule is then cut once with a restriction nuclease and used as a cloning vector for next DNA fragment.
What is the ultimate test for the function of a mutated gene?
Insert it into the genome of an organism and see the effect that it has. These are known as transgenic organisms, for example in mice.
Name some different types of microscopy
- fluorescence microscopy
- confocal microscopy
- light microscope for cells
