Molecular Biology Flashcards
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What is recombinant DNA technology?
Also known as Gene Cloning
Allows isolation and manipulation of DNA.
Utilizes restriction enzymes and cloning vectors.
What is a cloning vector?
A DNA molecule that can be introduced into a host organism and can self-replicate. An example would be plasmids.
How do restriction enzymes work?
They recognize specific nucleotide sequences in the DNA and cuts both strands of the sugar-phosphate backbone.
Different restriction enzymes cut different sequences, but when cut, the sequence always forms a palindrome.
The DNA fragments can have cohesive ends or blunt ends.
How do bacteria prevent restriction enzymes cutting the bacteria’s DNA?
Bacteria make both the restriction enzyme and a sequence specific methylase.
The Methylase adds a methyl group at the restriction site, so cleavage is blocked because the restriction enzyme can no longer bind to its restriction site.
What is agarose gel electrophoresis?
Agarose is a large carbohydrate polymer that forms a gel.
An electric current is applied across the gel, and DNA will move towards the positive electrode, because DNA is -vely charged. Smaller DNA will move faster than larger fragments.
Gel must be placed in a buffer to allow current to flow through it.
How do you do restriction enzyme mapping?
- DNA is cut using individual restriction enzymes, creating restriction maps (otherwise known as digested DNA)
- DNA is separated using gel electrophoresis
- DNA fragment size can be identified by comparing it to the known DNA fragments
- This allows you to find out how often each restriction enzyme cuts the DNA and the position of their sites relative to one another
- Different restriction enzymes are used in pairs to identify where their sites are, relative to one another
RFLP…
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What is the structure of plasmids?
Origin of replication (ori), which allows replication independent of bacterial chromosome.
AB resistance gene (AB= antibiotic)
MCS (Multiple Cloning Sequence)
How is the recombinant DNA amount amplified?
After the DNA fragment has been inserted into the plasmid, the plasmid is taken up by E.coli.
Using the ori, 100-200 copies of the recombinant plasmid is made.
As bacteria divide to form colonies, Each bacteria will contain 100-200 copies of the recombinant plasmid.
How are plasmids engineered for DNA cloning?
They contain the MCS. This is a cluster of restriction sites, which allows different restriction enzymes to cut the same plasmid.
Additionally, MCS of many plasmids are placed within a lacZ gene, which is useful in identifying recombinant plasmids.
What are cosmids?
Modifies plasmids vectors that are present at a low copy number that can hold 30-40kb cloned DNA.
Plasmids with insertes over 20kb are difficult to maintain.
How is eukaryotic DNA used as recombinant DNA?
Eukaryotic genes have a lot of bases due to the large amount of introns. So to reduce the size of the genes in order for them to fit on the plasmids, the mRNA of the gene is used.
The mRNA does not contain the introns, so DNA can be synthesized from it to form cDNA, which is used in recombinant DNA.
What is cDNA?
Complementary DNA.
It is DNA synthesized from mRNA that contains no introns. cDNA is produced using reverse transcriptase enzyme.
What are cDNA libraries?
Collection of cDNA copies (in the form of plasmids) of the mRNA present in a cell population and represent the genes being expressed in the population from where mRNA is harvested.
What are expression vectors?
Plasmid cloning vectors designed to allow expression of cloned genes in bacteria, with the purpose of producing large quantities of encoded protein.
What are deoxynucleotides?
dNTP
Nucleotides which have a pentose sugar missing a -OH group on the 2’ carbon.
What are dideoxynucleotides?
ddNTP
Nucletides which have a pentose sugar missing two -OH groups on the 2’ and 3’ carbons
What is radioactive dideoxynucleotide sequencing?
AKA original Sanger sequencing.
DNA is replicated by DNA polymerase in the presence of dNTP, a small amount of ddNTP and a radioactive tracer (35S -> Sulphur isotope).
Some fragments end prematurely and the length of the fragment depends on when ddNTP is incorporated.
Sequencing is done 4 times, each time with a different ddNTP; 1) dNTP + ddATP; 2) dNTP + ddCTP; 3) dNTP + ddGTP; 4) dNTP + ddTTP
The products from each of the 4 sequencing reaction are run in separate lanes, prepared gel electrophoresis. The fragments separate based on size and are identified by the radioactive isotope.
The shortest bands are the DNA products closest to the primer, so travel faster and further on the gel. The gel is read from the bottom up to sequence the DNA.
What is automated DNA sequencing?
AKA fluorescent capillary sequencing.
A single reaction for each DNA sequence is used, which includes all four ddNTP. Each ddNTP is labelled with a unique fluorescent marker.
The fluorescent label on each ddNTP has a different wavelength. As the fragments move down the gel, it passes a laser, which excites the fluorescent marker. The wavelength of the fluorescence is read as the fragment passes.
A digital interpretation of the sequence derived from the wavelength of fluorescent emissions is produced.
What is the issue with large scale automated DNA sequencing?
The resolution of the produced wavelength will become more and more unreadable as the size of the DNA sequenced increases.
How is large scale sequencing of DNA done?
Fragment DNA into small pieces. Clone into vector. Sequence DNA clones using primer in vector. Use mapping data and sequence overlap between clones to align sequence from fragments to get complete sequence.
