Molecular biology Flashcards
Explain the 5’ - 3’ / 3’ - 5’ directionality concept.
Directionality is the orientation of a single nucleic acid NA strand.
In a strand, one end contains a PO4 on the 5th C of a pentose, and the other end contains an OH on the 3rd C of the pentose.
Palindrome
dsDNA with the same sequence on both complementary strands
Does the base pairing have to be perfect for primer-dimer formation?
No
are nucleic acids in solution linear?
NA are usually base-paired in solution. Either intra- or inter-molecularly.
What is the difference between DNA and RNA pentoses?
Why is DNA called “deoxyribonucleic”?
DNA contains an H on the 2nd C of the pentose
RNA contains an OH (hydroxil).
The 2’ group in DNA is deoxygenised (i.e. deoxyribonucleic acid).
Wha it is the structural base of all nucleic acids?
What is a nuceloside?
What is a nucelotide?
NTP (nucleoside triphosphate), i.e. 3 phophates and a nuceloside.
A nuceloside is a pentose + nitrogenous base.
A nucelotide is a pentose + nirogenous base + one phosphate group.
Briefly explain RNA alkaline hydrolisis.
Phosphodiester bonds are broken by OH-
Three requiemens for DNA synthesis?
1) base-pairing at the 3’ end
2) 3’ must have an OH
3) ssDNA template downstream of this site
What is an oligonucleotide?
A short NA sequence (note: a primer is an oligonucelotide)
Briefly explain DNA acid hydrolisis.
1) excess H+ attacks N7 of A-G
2) O- in breaks phosphodiester bond
What does DNA and RNA stand for?
deoxyribonucleic acid and ribonucleic acid, respectively
Chargaff’s rule
- DNA should have a 1:1 stoichiometric ratio of purine and pyrimidine bases
- the amount of G should be equal to C and the amount of A should be equal to T
Polarity of nucelcic acids
Nucelic acids are hydrophobic, thus, they are more likely to bind with each other than with water. Consequently, in solution they bind together.
B form of DNA: definition
Double-stranded, right-handed with antiparallel strands.
The most biologically important form of DNA.
B form DNA: structural description
- *1)** Contains major and minor grooves.
- *2)** distance between adjacent bases is 0.34 nm.
- *3)** 1.9 nm in diameter.
- *4) 10** bases per complete turn.
Major grooves in DNA: definition and biochemical importance
Definition: Portions in which backbone is widely separated.
Biochemical importance: They allow protein binding
Major grooves in DNA: definition and biochemical importance
Definition: Portions in which backbone is widely separated.
Biochemical importance: They allow protein binding
Chargaff’s rule
- DNA should have a 1:1 stoichiometric ratio of purine and pyrimidine bases
- the amount of G should be equal to C and the amount of A should be equal to T
Protein - DNA interactions: mechanism
- exocyclical groups (i.e. gorups outside the pentose) recognized by protein
- sequence-specific
Double helix: bonding
Covalent:
1) Phosphodiester bonds: betwee pentoses; forms sugar-phosphate backbone
2) Glycosidic bonds: between bases and pentoses
Non-covalent:
3) Hydrogen bonds: between paired bases
4) Base-stacking: between adjacent bases
5) Shell of hydration: between phosphodiester backbone and water; stabilizes double helix and increases solubility
Differnces between A RNA and B DNA
RNA (A-form):
1) major groove is narrow and deep.
2) more compact than DNA B form
3) 11 bases per complete turn.
4) U instead of T
5) Can occasionally have G-U pairs
Pseudoknots
Pseudoknots are secondary structures in RNA characterized by having at least two stem loops
RNA secondary stuctures
RNA almost always forms secondary sructures, making it structurally similar to proteins
RNA has to unfold for translation
Pseudoknots
Pseudoknots are secondary structures in RNA characterized by having at least two stem loops
Chaperones and RNA
Chaperones are proteins that alter RNA structure and they are very important for RNa functionality
B form DNA: structural description
- *1)** Contains major and minor grooves.
- *2)** distance between adjacent bases is 0.34 nm.
- *3)** 1.9 nm in diameter.
- *4) 10** bases per complete turn.
Types of nucleic acid duplexes and order in strength
RNA:RNA > RNA:DNA > DNA:DNA
DNA:RNA duplexes: function
Transcription and DNA replication
RNA:RNA duplexes: function
RNA:RNA duplexes regulate gene expression
miRNA (micro RNA): function and mechanism
1) silences gene expression
2) miRNA binds to a RNA gene
RNA editing: definition and three types
1) post-transcriptional alteration of RNA sequences
2) insertion, deletion, substiution
Antiparalellism
Two strands parallel to each other but runing in opposite directions
tmRNA: name, function/mechanism
1) transfer-messenger RNA.
2) translates into an amino acid sequence that targets an improper protein or mRNA for degradation
Chaperones and RNA
Chaperones are proteins that alter RNA structure and they are very important for RNa functionality
Antiparalellism
Two strands parallel to each other but runing in opposite directions
tmRNA: name, function/mechanism
1) transfer-messenger RNA.
2) translates into an amino acid sequence that targets an improper protein or mRNA for degradation
RNA editing: definition and three types
1) post-transcriptional alteration of RNA sequences
2) insertion, deletion, substiution
miRNA (micro RNA): function and mechanism
1) silences gene expression
2) miRNA binds to a RNA gene
RNA:RNA duplexes: function
RNA:RNA duplexes regulate gene expression
DNA:RNA duplexes: function
Transcription and DNA replication
Types of nucleic acid duplexes and order in strength
RNA:RNA > RNA:DNA > DNA:DNA
RNA secondary stuctures
RNA almost always forms secondary sructures, making it structurally similar to proteins
RNA has to unfold for translation
Differnces between A RNA and B DNA
RNA (A-form):
1) major groove is narrow and deep.
2) more compact than DNA B form
3) 11 bases per complete turn.
4) U instead of T
5) Can occasionally have G-U pairs
Double helix: bonding
Covalent:
1) Phosphodiester bonds: betwee pentoses; forms sugar-phosphate backbone
2) Glycosidic bonds: between bases and pentoses
Non-covalent:
3) Hydrogen bonds: between paired bases
4) Base-stacking: between adjacent bases
5) Shell of hydration: between phosphodiester backbone and water; stabilizes double helix and increases solubility
Protein - DNA interactions: mechanism
- exocyclical groups (i.e. gorups outside the pentose) recognized by protein
- sequence-specific
B form of DNA: definition
Double-stranded, right-handed with antiparallel strands.
The most biologically important form of DNA.
Polarity of nucelcic acids
Nucelic acids are hydrophobic, thus, they are more likely to bind with each other than with water. Consequently, in solution they bind together.
expanded central dogma
Most RNA is non-coding and is used in gene expression
mRNA: name and function
complementary to a DNA gene; transformed into protein by a ribosme
Why does DNA have T instead of U?
1) occassional degradation of C to U would be problematic
2) ocassional T-T are more easilly fixed than U-U
alcohol-salt precipiation: objective, mechanism, and options
1) to concentrate and puify NA.
2) alcohol removes shell of hydration and allows insoluble NA-salt formation
3) ethanol (higher volume needed for RNA) or isopropanol
Pellet washing: rationale, method, and mechanism
1) to remove salt from NA-salt complex because in interferes with futue procedures
2) the water in the water-alcohol solution removes the salt.
3) 70% ethanol for DNA and 80% for RNA
nucelic acid pellet drying and resuspension: method
pellet is air-dried and resuspended in Tris-EDTA
Tris-EDTA: use and benefits
1) used to solubilize nucleic acids
2) Mimics cell pH of 8 and removes Mg2+ to reduce nuclease activity
NH4 (amonium) acetate precipitation: advantages
1) inhibits enzyme actvity and removes dNTP’s
siRNA: name and function
1) small interfering RNA
2) degrades a target mRNA after transcription to avoid translation
rRNA: name and function
1) ribosomal RNA
2) catalyzer for protien synthesis
tRNA: name and function
1) transfer RNA
2) link between mRNA and protein duting translation
snRNA: name and function
1) small nuclear RNA
2) processes pre-messenger RNA
snoRNA: name and function
1) small nucleolar RNA
2) modify oter RNA chemically
Ribozymes: definition and funtion
1) catalytically active RNA molecules
2) similar function to enzymes
miRNA: name and function
1) micro RNA
2) regulates translation
Denaturation: definition
Denaturation aka melting is the separation of DNA strands.
Tm: definition
The temperature at which half of the dsNA molecules in a mix denature (i.e. separate to ssNA). Tm is different for each NA molecule.
Denaturation: definition
Denaturation aka melting is the separation of DNA strands.
What factors determine Tm?
[GC]; sequence lenght; [salt]; [organic solvents]; pH; T°
Does denatuation depend on [DNA]?
[DNA] doesnot affect denaturation because it’s a unimolecular reaction. The separation of one molecule does not influence the separation of another one.
What is NA renaturation (hybridization)?
It’s the formation of dsNA from ssNA.
What do we aim to accomplish when renaturating DNA?
We aim for perfect base pairing.
What are the steps of renaturation?
1) Nucleation: short complementary regions form when ~3bp complement each other
2) Zippering: when adjacent bases also complement each other, rapid base-paiting occurs.
What do we aim to accomplish when renaturating DNA?
We aim for perfect base pairing.
What is NA renaturation (hybridization)?
It’s the formation of dsNA from ssNA.
Does denatuation depend on [DNA]?
[DNA] doesnot affect denaturation because it’s a unimolecular reaction. The separation of one molecule does not influence the separation of another one.
What factors determine Tm?
[GC]; sequence lenght; [salt]; [organic solvents]; pH; T°
Define annealing stringency.
Stringency can be explained as the strictness of Watson - Crick pairing (i.e. close to perfect base pairing).
How would you ensure annealing stringency?
1) raising temperature
2) lowering [salt]
How do you usually know the Tm of an oligonucleotide?
Usually you don’t use a spectrophotometer, but instead you use a formula.
When you order an oligonucleotide, it comes with instructions and its specific Tm, which sometimes is accurate but not everytime.
1) If - no product, the Tm was too high.
2) If - too much product, the Tm was too low.
There’s a simplified formula for DNA-DNA duplexes.
When can it be used?
Tm = 69.3 + 0.41 (%G + C)
Can be used for 0.3M Na+ in the absence of formaldehyde (organic).
Why is this called a 5’ overhang?
Because the overhang (long pairless sequence) is on the 5’ end.
What are the steps in an entire PCR thermocycling reaction?
1) Initial denaturation : 94 °C / 5 min
2) Step cycling as described before but 20 - 30 cycles
3) Final extension: 72 °C 1 - 10 min to ensure that all products are full lenght
4) Optional: Set the thermocycler to hold a (4 °C nothing happens in this step).
What are the causes for amplificaiton plateuing during the final cycles of PCR?
1) Lack of substrate (i.e. reduction in primer concentrations)
2) DNA pol degradation
3) dNTP degradation because of high temepratures
4) Accumulation of primer-dimers
5) Products can start annealing to each other.
What do we aim for when optimizing PCR?
What are the factors to consider for optimizing PCR?
We aim to avoid false priming at low temperature
The factors to consider for PCR optimization are:
1) hot start
2) primer design
3) annealing temperature
4) PCR components
5) PCR controls
6) Zero exogenous DNA contamination
When binding primers to target DNA, why do most problems occur at 3’ ends of primers?
Most problems occur at the 3’ ends of primers because they need perfect complementarity to allow DNA synthesis.
Does the primer have to match the target DNA sequence perfectly?
The primer sequence does not have to match the target DNA sequence perfectly, except at the 3’ end.
Note that primers do not have 5’PO4 unless added
How can annealing temperature be opimized for PCR?
1) trial and error: trying a range of different annealing temperatures
2) adding Dimethyl sulfoxide (DMSO) 3 - 6%.
3) perform touchdown PCR
4) perform gradient PCR
[Mg2+] is the most important component in a PCR reaction.
- *1)** In what quantity is it used?
- *2)** What is its chemical function?
- *3)** What happens if [Mg2+] is too low?
- *4)** What happens if [Mg2+] is too high?
1) Mg2+ is usually used at 1 - 3 mM
- *2)** Mg2+ binds to dNTP’s and forms Mg-dNTP, which is the complex used for synthesis by the DNA pol
- *3)** Low [Mg2+] increases specificity
- *4)** High [Mg2+] decreases specificity (stabilizes shell of hydration too much) and results in non=specific products and wrong DNA synthesis. This results in smeared gel (see image).
Broadly, how does PCR annealing temperature optimization work with touchdown PCR?
The functional Tm’s of the primers are not known. Touchdown PCR allows the first DNA synthesis at the highest temperature possible (i.e. the closest temperature to Tm as possible), at which you should get 100% annealing.
