Molecular Genetics 1-6 Flashcards
What and when was the Hershey-Chase experiment?
1952
Involved the use of a phage (virus) which affects E. coli. The phage could replicate once inside E. coli cells. In one experiment they labelled the protein with radioactive sulphur, and in the other they labelled the DNA with radioactive phosphorus. Then they allowed the phage to infect E. coli and centrifuged the E. coli cells. When the protein was labelled, radioactivity was only found in the liquid, not pellet. Radioactivity was found in both when DNA was labelled
When did Watson and Crick propose the structure of DNA?
1953
Who helped Watson and Crick come to their conclusions on DNA structure?
Rosalind Franklin and PhD student Raymond Gosling
How are the carbons labelled in a DNA molecule?
5’ carbon at phosphate end
3’ carbon has hydroxide group
Which nucleotide bases are purines?
Adenine and guanine
Which nucleotide bases are pyrimidines?
Cytosine and thymine
What is a gene?
The basic unit of inheritance by which hereditary characteristics are transmitted from parent to offspring
What is a gene at the molecular level?
A length of DNA (or in some viruses RNA) which exerts its influence on the organism’s form and function by encoding and directing the synthesis of a protein, or a tRNA, rRNA or other structural protein.
In eukaryotes, a gene also includes noncoding introns
What is the name for the type of mRNA produced in prokaryotic transcription and what does it mean?
Polycistronic
A single strand of mRNA that encodes several proteins
What is the purpose of the 5’ cap of mRNA?
The cap plays a role in the ribosomal recognition of messenger RNA during translation into a protein
What enzyme places a 5’ cap on the end of the mRNA molecule?
Gaunyl transferase
What is the poly A tail at the 3’ end of the mRNA molecule?
It is a string of adenine bases. It’s addition is catalysed by the enzyme poly (A) polymerase
Where is DNA found? (4 places)
Nucleus
Cytoplasm
Plastid
eDNA (environmental DNA)
In what direction is mRNA transcribed?
In a 5’ to 3’ direction along the coding strand, against the direction of the template strand
What is eDNA?
Found in soils, water, faeces, etc
Useful for ecological surveying - what species are in the area
DNA in our food can test if packaging is honest about contents
How is prokaryotic DNA packaged?
- Arranged into nucleoids
- Supercoiled with the help of architectural proteins
- Usually circular DNA genome with nonessential genes in plasmids
- Eukaryotic organelles resemble prokaryotes in their genomic organisation
How is eukaryotic DNA packaged?
- Packaged into linear chromosomes
- Chromatin - DNA supercoiled using histones
- Nucleus is membrane-bound
- Some eukaryotic genomes organised into operons
How are chloroplasts inherited?
Through the cytoplasm - maternally inherited
Steps of plasmid inheritance
- F plasmid carries genes necessary for conjugation
- Plasmid may have additional genetic information from chromosome
- One strand of F+ cell plasmid DNA breaks at arrowhead
- Broken strand peels off and enters F- cell
- Donor and recipient cells synthesise complementary DNA strands
- Recipient cell now also has copy of F plasmid
Prokaryotic DNA replication
- The origin of replication (ORI) is where replication begins
- Replication forks in both directions form, leaving two daughter molecules
Eukaryotic DNA replication
- Multiple ORIs, multiple replication forks and bubbles
2. Two daughter DNA molecules formed
Role of topoisomerase in DNA replication
Breaks, swivels and rejoins the parental DNA, ahead of the replication fork, relieving the strain caused by unwinding
What is the role of helicase in DNA replication?
Unwinds and separates the parental DNA strands
What is the role of primase in DNA replication?
Synthesises RNA primers, using the parental DNA as a template
What is the role of DNA polymerase I in DNA replication?
Removes the RNA primer and synthesises DNA fragments in its place
What is the role of DNA polymerase III in DNA replication?
Synthesises new DNA strand in 5’ to 3’ direction
What is the role of single-strand binding proteins in DNA replication?
Stabilise the unwound parental strands
Which DNA strand is easy to replicate?
The leading strand, as this strand is being replicated in the 5’ to 3’ direction
Which strand is harder to replicate and why?
The lagging strand is harder to replicate as it runs in 3’ to 5’ direction. Primase must produce many RNA primers and hop along placing primers periodically along the DNA
What are the four main steps of DNA extraction?
- Cell lysis and protein removal
- Precipitation
- Wash
- Resuspension (or ELUTION)
Cell lysis - why do you need it?
Lysis of the cell membrane is required to release the DNA
What is in a cell lysis buffer?
- Detergent (e.g. SDS, an an ionic surfactant) - to help break up fats and open up cells
- Salt (e.g. NaCl) - to help break down hydrogen bonds between DNA strands, enabling it to dissolve in the solvent
- EDTA - chelation of divalent ions, which prevents DNases from working
- Tris - acts as a buffer to stabilise alkaline pH
Why would you want to remove proteins during DNA extraction?
DNA is packaged in proteins, so this must be removed. You may also want to remove any contaminating cellular proteins
What happens to proteins when exposed to the lysis buffer?
They are denatured and dissolved by the lysis buffer
What other chemicals assist in protein removal?
- Proteinase (e.g. proteinase K)
- Chaotropic salts (e.g. guanidine hydrochloride) - weaken the forces holding the protein together, thus leading to denaturation
- Ammonium acetate - this will cause proteins to precipitate, so you can centrifuge the solution and remove unwanted proteins which will form pellet
What is precipitation in DNA extraction?
Separating the DNA from the proteins by DNA precipitation
What is added to precipitate the DNA and how is it physically separated?
Isopropanol or ethanol - DNA is insoluble in both of these chemicals, so it leads to the aggregation of DNA molecules out of solution. DNA can then be separated by centrifugation or with a silica matrix in a column
Why must the DNA pellet or matrix be washed?
To remove contaminants
How is the pellet washed?
With 70% ethanol - the salts you are trying to remove will not dissolve in 100% ethanol
How is resuspension carried out?
DNA is soluble in water, so most DNA preps will use purified water to re-suspend the pellet of DNA or to pass through the matrix and collect the DNA elute in a tube.
Another popular resuspension solution is TE buffer, which includes Tris and EDTA
What is in vivo DNA synthesis?
DNA replication within a cell
What is in vitro DNA synthesis?
DNA produced in a test tube/lab
What are the building blocks for synthesising DNA in nature?
Deoxyribonucleoside triphosphates (dNTPs). As the new base gets added to the 3’ end of the chain, two of the phosphates are lost
What are used instead of dNTPs when building DNA synthetically (without the use of enzymes)?
Phosphoramidites
Steps of building a synthetic DNA chain
- Detritylation removes the DMT blocking groups so that the next base can be added
- Phosphoramidites are sequentially added to the column to build up the sequence of choice by a coupling reaction
- Capping occurs to block unreacted OH groups from receiving new
- Oxidation is induced with iodine to stabilise the phosphodiester bond
What is synthetic DNA used for?
Primers, probes, building synthetic genomes or storing digital data
In what direction is DNA synthesised when produced synthetically?
3’ to 5’ direction
When was DNA polymerase first purified?
1958
Who developed the early PCR protocol in the 1960s and 70s?
Khorana and Molineaux
How long would it take an experienced post doc to synthesise primers 10 bases long?
0.5-2 years
What was the issue with early PCR techniques
The polymerase wasn’t thermostable so had to be added again after each cycle
What is the name of thermostable polymerase?
Taq polymerase
When we’re modern PCR protocols with Taq polymerase developed?
1985
Who won the Nobel prize for the discovery of Taq polymerase and when?
Kary B. Mullis in 1993
What does the modern PCR reaction require?
Primers Taq polymerase dNTPs DNA template Buffer
What thermophile was Taq polymerase isolated from?
Thermus aquaticus
What are the three temperatures of the PCR cycle and what happens at each temperature?
1) 94 degrees Celsius: to break hydrogen bonds and separate double strands
2) 60 degrees Celsius: to allow complementary primers to anneal to DNA
3) 72 degrees Celsius: optimum temperature for Taq polymerase to extend the DNA chains
What happens to the DNA primers after PCR?
They are incorporated into the strand, they are not removed
What must be added to PCR products for next generation sequencing?
Labels
What enables easy cloning of PCR products into a vector?
Restriction sites being added onto the ends
How can point mutations be induced in PCR?
By using a mismatched primer, differing in only one base from the DNA strand. Wild type and mutant plasmids are produced this way
What selectable markers can be used to distinguish between mutated and wild type plasmids?
Antibiotic resistance
Remove a restriction site of an ending lease (and treat the plasmid with this enzyme prior to the transformation)
What is selection?
The process of selecting the bacterial cells which have taken up a plasmid
What antibiotics do plasmid resistance genes usually make the bacteria resistant to?
Ampicillin or kanamycin
What gene confers resistance to ampicillin?
bla - beta lactamase gene
What is a multiple cloning site?
A DNA region within a plasmid that contains multiple unique restriction enzyme cut sites
What is an operon?
An area holding several genes with similar functions e.g. lactose metabolism
What does the lacZ’ gene encode?
A protein called B-galactosidase, which is involved in breaking down lactose
What represses the expression of the lactose metabolism operon when lactose is not present?
A lac repressor
How does lactose allow the expression of the genes within the operon?
It interferes with the binding site and represses the repressor, leaving RNA polymerase free to undergo transcription and translation, producing the proteins
What is the molecular analogue to lactose?
IPTG
What is the alternative substrate to lactose given to B-galactosidase?
X-gal
What is the colour signal when B-galactosidase has broken down X -gal?
Colony changes from white to blue
What are the two parts that B-galactosidase can be split into that work only when together?
a-peptide (can be put on plasmid)
w-peptide (in chromosome)
What is the name of the mutant when only the w-peptide is present in DNA?
lacZ🔼M15 mutant
What is the difference between the mutant and wild type E. coli?
Wild type has intact lacZ’ gene so produces intact B-galactosidase, so when grown on a medium of IPTG and X-gal, colonies will look blue. On the other hand, the mutant only produces the w-peptide of B-galactosidase, so colonies will look white
What plasmid has the lacZ’ gene in it?
pUC18
When the E. coli mutant is transformed with a non-recombinant pUC18, what colour is the colony when grown on an X-gal medium?
Blue - lacZ’ gene not disrupted by restriction enzyme
When the E. coli mutant is transformed with a recombinant pUC18, what colour is the colony when grown on an X-gal medium?
White - lacZ’ gene disrupted but restriction enzyme and gene inserted
What is the difference between a deoxyribose sugar and a divide sugar?
Deoxyribose sugar has single -OH group
Ribose sugar has two -OH groups
What does ddNTP stand for?
dideoxyribonucleoside triphosphate
What does a ddNTP lead to chain termination?
No -OH group on sugar, and there must be one to continue chain
Other names for Sanger sequencing
Chain termination sequencing
Dideoxy-sequencing
How does Sanger sequencing work?
1) Oligonucleotide primer anneals to single strand DNA. The DNA strand is extended by DNA polymerase with a mixture of dNTPs and ddNTPs
2) Thermal cycling similar to PCR
Process of chain termination sequencing using ddNTPs
1) Fluorescently label ddNTPs (e.g. one colour for ddA, another for ddT, etc.)
2) Add DNA polymerase
3) Add ddGTP in low concentration
4) dATP, dGTP, dCTP and dTTP
5) Run through gel electrophoresis
Three ways to measure genetic diversity:
1) Morphological traits: growth habit, weight at maturity, size, shape, etc. However traits can be affected by environment
2) DNA sequencing: expensive
3) Molecular markers (genotyping): cheaper, faster
What are molecular markers?
Pieces of DNA which occur randomly throughout the genome that have been shown to be associated with a particular trait
What is important about molecular markers?
They vary in form when a different allele is present for that particular trait
What makes a good molecular marker?
One that can distinguish between homozygotes and heterozygotes
Example of molecular marker
Microsatellites
What are the other names for microsatellites?
- Simple sequence repeats (SSRs)
- Sequence tagged simple sequence repeats (STSSRs)
What is the occurrence of producing microsatellites?
Error made whilst replicating microsatellite DNA every 10-20 generations
Why do microsatellites exist?
Some repeats may from Z-DNA, which may play a role in binding certain regulatory proteins, or recombination
How many microsatellites does the average chromosome contain?
700-1000
How are microsatellites used?
Surrounding a microsatellite is a unique sequence that appears nowhere else in the DNA of that species. Primers can be created for this unique DNA, so only that specific microsatellite will be amplified. Fragment size varies based on the length of the repeat
Why might there be more than one band on the gel when a microsatellite DNA sample is amplified?
DNA polymerase made a few mistakes during PCR and this led to fragments with more or less bases
What is the cost of using a microsatellite assay instead of sequencing?
0.5p, 1/1000th of the cost of sequencing
Benefits of simple sequence repeats
- SSRs are single locus, codominant, multi-allelic and PCR-based molecular markers
- Can be used in mapping and diversity studies
- SSRs can be multiplexed and automated
Why is being co-dominant a benefit of using SSRs?
Can distinguish between heterozygotes and homozygotes
Why is being multi-allelic a benefit of using SSRs?
Can determine specifically which of many alleles is present
What do multiplexed and automated mean?
Can take microsatellites and primers from an allele on one chromosome, put them in the same PCR machine, run them on the gel and allow the gel to distinguish between the two microsatellites using fluorescent markers and automated sequences
What is the problem with most molecular markers?
Need human intervention, and data analysis is time-consuming
What is an SNP (single nucleotide polymorphism)?
The simplest and most common type of genetic variation, found every 1000 base pairs along the human genome
What is an example of a disease caused by an SNP?
Sickle-cell anaemia - a single base substitution from A to T which changes one amino acid from glutamate to valine at position 6 of the protein. Red blood cells sickle-shaped
Which SNPs are more common than others?
C to T is far more common than C to A.
A to G is more common than G to T.
It is easier to swap a purine with a purine than a purine with a pyrimidine, which could change the tertiary structure of the DNA
Method 1 to convert a SNP into a useful molecular marker
PCR-RFLP:
Use a restriction enzyme to cut the DNA. If there is a SNP, the DNA will not be cut into two pieces.
What is the problem with PCR-RFLP?
A machine may wrongly process pieces which have not been cut and give a wrong result
Method 2 to convert a SNP into a useful molecular marker
Allele specific oligonucleotide (ASO) probe - detection of globin mutation:
Two probes are designed, one complementary to the normal DNA sequence and the other complementary to the nucleotide DNA sequence. Probes can be labelled with two different fluorescent markers. Add primers and DNA to thermal cycled. Heat up DNA so it denatures and immobilise it on an insoluble matrix. Allow it to cool so probes can find their complementary sequences. Once your probes have hybridised you can wash away the rest of the mixture then assay for the presence of probes. If the probe hybridises, you will see fluorescence
How many DNA samples fit in one assay?
35,000
What is the name for when multiple probes are used for different regions of DNA at the same time?
Multiplex assay
Advantages of SNPs
Cheaper than sequencing (1/1000th of cost) Most frequent form of DNA variations Abundant and have slow mutation rates Easy to score via automation High throughput
