Molecular Genetics (Rose Murray 1-4) Flashcards
Whose experiments concluded that DNA is found inside cells and not proteins?
Hersey and Chase (1952)
How did Rosalind Franklin (& Raymond Gosling) help Watson and Crick make their famous discovery?
produced the first x-ray diffraction image of DNA
Name the Pyrimidines
Thymine, Cytosine and Uracil
Name the Purines
Guanine and Adenine
How does alternative splicing provide phenotypic variation?
some introns may be left, coding for a different protein from the same gene.
3 main differences between eukaryotic and prokaryotic translation?
- Eukaryotic transcripts leave the nucleus before translation, whereas in prokaryotes, translation occurs right away.
- Eukaryotic transcripts have a polyA tail and a 5’ cap, whereas prokaryotes have neither.
- Single gene per eukaryotic mRNA transcript, however, prokaryotic transcripts can be polycistronic.
What is eDNA?
Environmental DNA, found in soil, water, faeces, air etc
3 points about prokaryotic DNA packaging
- Arranged in nucleoids (not membrane-bound organelles)
- Supercoiled around architectural proteins (not histones)
- Singular, circular DNA with non-essential genes on plasmids in cytoplasm
3 points about eukaryotic DNA packaging
- Packaged into chromosomes
- Supercondensed around histones
- located in the nucleus
What is significant about the inheritance of chloroplast DNA (cpDNA)?
The type of egg cell determines the type of chloroplast.
Experiment: Mirabilis plants
eggs cell determined whether chloroplasts were white, green or both (variegated). Sperm had no effect.
Which plasmid can the genes for conjugation be found?
F plasmid
it can pop in and out of the chromosome by homologous recombination.
What is the role of Topoisomerase in the initiation of DNA replication?
relieves the tension of wound up DNA by breaking and rejoining it ahead of the replication fork.
What is the role of Helicase in the initiation of DNA replication?
Unwinds and separates the DNA strands
What is the role of Primase in the initiation of DNA replication?
makes RNA primers by using the DNA as a template.
What is the role of DNA polymerase III in the initiation of DNA replication?
synthesises new DNA from the 5’ to 3’ direction.
What is the role of DNA polymerase I in the initiation of DNA replication?
on the lagging strand, removes the RNA primers and synthesises DNA fragments in its place.
What is the role of DNA ligase in the initiation of DNA replication?
joining of the fragments
In DNA extraction, what is the role of detergents?
To break up fats/lipids so the membrane opens.
In DNA extraction, what is the role of salts?
to weaken the H bonds between DNA strands.
In DNA extraction, what is the role of EDTA?
grabs ions with a valency of 2 (can make 2 bonds) and prevents DNAses from working.
In DNA extraction, what is the role of Tris?
a buffer to stabilise an alkaline pH.
In DNA extraction, what is the role of isopropenol or ethanol?
DNA is insoluble in them so it precipitates out.
It can then be caught by a silica matric in via a spin column.
Ethanol also used to wash out salts.
In synthetic oligonucleotide synthesis, what are used insted of dNTPs?
Phosphoamidites
they’re sequentially added to the column to build up a chosen sequence via a coupling reaction. The chain is grown from 3’ to 5’, opposite to normal.
What are the ingredients of a PCR mix?
dNTPs, Primers, Buffer, DNA template and Taq polymerase
What are the three stages of PCR and their respective optimum temperatures?
Denature strands - 94°C
Annealing of primers - 60 °C (depends on the primer)
Extension -72 °C
How can PCR be used to enable easy cloning into vectors?
Add restriction sites to the ends of the PCR product
What two antibiotics are most bacterial vectors resistant to?
Ampicillin and Kanamycin
What does the LacZ gene code for?
beta-galactosidase enzyme
How does lactose regulate the Lac operon?
The Lac I gene is always switched on, producing a repressor molecule which binds to the regulatory region, preventing RNA polymerase from transcribing the rest of the operon. If lactose is present, it binds to the repressor, inhibiting it and the Lac operon is expressed, producing beta-galactosidase.
How can we use the LacZ gene to screen for recombinant bacteria?
we can culture the colonies on IPTG or X-gal, which replaces the function of lactose in the pathway. Wild-type, non-recombinant bacteria will produce beta-galactosidase and X-gal will be broken down into galactose and a blue colour.
The LacZ∆M15 mutants (recombinants) will have taken up the DNA, interrupting the LacZ gene, preventing its translation. As a result, these colonies will grow white and can be selected.
In sequencing, what is the difference between dNTPs and ddNTPs?
dNTPs have only lost one OH group so can still make connections with other nucleotides.
ddNTPs have lost both OH groups so can no longer make connections and terminates the chain.
Briefly, explain Sanger Sequencing
PCR is carried out with dNTPs and ddNTPs (e.g. ddGTP) everytime a ddNTP is incorporated, the chain terminates. This produces different lengths of DNA which can be analysed by gel electrophoresis. The ddNTPs can also be fluorescently probed with different colours and automated process can produce a chromatograph.
