DNA Technology Flashcards
1
Q
What is the pentose sugar found in nucleotide made up of?
A
5 carbons that form a cyclical structure with oxygen
Nitrogenous base joined to C1
Phosphate group joined to C5
Hydroxyl group joined to C3
2
Q
Which nucleotides are pYrimidines?
A
CYtosine + thYmine (+ uracil)
3
Q
Which nucleotides are purines?
A
Adenine + guanine
4
Q
How many hydrogen bonds between cytosine and guanine?
A
3
5
Q
How many hydrogen bonds between adenine and thymine?
A
2
6
Q
How are sugar phosphates linked?
A
By phosphodiester bonds
7
Q
Base stacking?
A
Hydrophobic interactions -> arrangement of bases set above each other internalised to the structure (+ excludes water)
8
Q
VdW forces?
A
Individually small but contributes to stability
9
Q
What does double-stranded DNA form?
A
Two antiparallel strands with negatively charged phosphates on the outside
10
Q
What happens when DNA is denatured?
A
Double stranded molecule is converted to single stranded molecule
H-bonds within double helix are disrupted
11
Q
How is DNA denatured?
A
When DNA in solution is heated or using a strong alkali/urea
12
Q
How can denaturing be measured?
A
Optically by absorbance at 260nm. Absorbance of light increases at 260nm.
13
Q
What is hyperchromicity?
A
Increase of absorbance of a material
14
Q
What is the melting temperature (or Tm)?
A
Point at which 50% of al strands separate
15
Q
What is Tm dependent on?
A
GC content Length of DNA molecule Salt conc. pH (alkali is denaturant) Mismatches (unmatched bps)
16
Q
Tm + GC content?
A
Higher GC content = more H-bonds = higher Tm
% GC = (G+C)/(G+C+A=T) X100
3 H-bonds in GC vs 2 in AT
17
Q
Tm + molecule length?
A
The longer the continuous duplex, the more H-bonds, the greater the stability, the higher Tm.
However little further contribution after 300bp
18
Q
Tm + salt conc.?
A
Salt stabilises DNA duplex, so high [Na+] = high Tm
Increasing salt conc. overcomes the stabilising effect of mismatched bps, reducing the specificity of base pairing
19
Q
Tm + pH?
A
Chemical denaturants disrupt H-bonds NaOH ⇌ Na+ + OH- OH- disrupts H-bond pairing Few H-bonds = lower Tm High pH (alkalinity) destabilises DNA duplexes
20
Q
What is a mismatch?
A
A bp combination that is unable to form H-bonds
21
Q
Tm + mismatches?
A
Reduced number of H-bonds = lower Tm
Shorter continuous of double-stranded sequence = lower Tm
Mismatches also distort the structure + destabilises adjacent base pairing
22
Q
What is the reversal of denaturation?
A
Renaturation
Formation of structure favours energy minimisation
23
Q
What is renaturation facilitated by?
A
Cooling + neutralisation
24
Q
What is hybridisation?
A
Formation of duplex structures of 2 DNA molecules that have been introduced to each other (e.g. involving a primer)
25
Complementarity + Tm?
Basis of specificity
Perfect matches have higher Tm + are thermodynamically favoured over mismatches
Can use this property to form complementary molecules with no mismatches
26
Hybridisation + Northern blotting?
Analysis of mRNA (is a gene expressed?)
Limited technique as only detect one gene at a time + small numbers of samples
Largely superseded by quantitative PCR + microarrays
(Southern blotting is similar by analysis is of DNA)
27
What are other nucleic acid based techniques involving hybridisation?
Microarrays
Sequencing
PCR
Cloning
28
What is a probe?
Fragment of ssDNA (or RNA)
20-1000 bases in length
Complementary to the gene sequence of interest
Labelled with fluorescent/luminescent molecule (less commonly a radioactive isotope)
29
What is stringency?
Manipulating conditions - limiting hybridisation between imperfectly matched sequences increases specificity
High stringency - only complementary sequences hybridise
Temp near Tm
Low salt conc.
30
When are microarrays used?
To analyse expression of thousands of transcripts in each sample
31
Microarrays in Northern blotting?
Samples are fixed to solid surface (membrane) + a single probe is hybridised to a gene of interest
32
How are probes used in microarrays?
Ordered assembly of thousands of nucleic acid probes fixed to a solid surface
The sample of interest is hybridised to the probes - unique portion of mRNA transcript hybridises to a probe
Lots of copies of the same probe in a 'spot'
33
What do 'spots' indicate in microarrays?
Lots of 'spots' = detection of lots of transcripts
Bright 'spot' = high expression level
Faint 'spot' = low expression level
34
What is gene expression profiling?
Simultaneously measure 50,000 different transcripts (in cell/tissue/organ)
35
Microarrays + SNPs?
Microarrays can assess millions of SNPs (up to 2.5mil simultaneously)
Used in association studies (e.g. gene association with disease)
36
What is PCR?
Enzyme-based method to specifically amplify DNA segments using thermal DNA polymerase in a cyclical process
37
How does DNA polymerise synthesise a new strand?
By making a complimentary copy of the opposing template strand
Enzyme recognises a specific structure consisting of partially stranded DNA forming an initiation complex with it
38
How is the partially doubled-stranded structure formed?
By annealing a single-stranded primer to a denatured (thus single-stranded) template molecule
39
What is annealing?
Essentially hybridisation of a primer
| Results from formation of bps, stabilised by H-bonding between purines + pyrimidines
40
Which is usually chemically labelled whilst the other one is not: a probe of a primer?
Probe is chemically labelled
41
What are the primer reaction mixture concentrations?
Low copy number (low conc.) of template
Huge excess of primer which drives the reaction
Preferential annealing of the primer occurs
42
What is DNA dependent DNA polymerase?
Enzyme used in PCR, synthesises a new nucleic acid strand by copying DNA molecule
It cannot copy RNA or make RNA
RNA must first be copied to DNA by reverse transcriptase before it can be amplified by PCR
43
What does DNA dependent DNA polymerase require?
Template strand with a primer (usually 20-30 bases) annealed to it
Deoxy-nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
3' end available for extension, requires 5' overhang
44
What is a chain reaction?
Series of events, each one dependent upon the preceding event
Leads to exponential increase in number of events occurring
Each cycle doubles the number of molecules present
45
Where does specificity stem from?
Primer complementarity
46
Amplification of DNA segments?
Segment amplified is determined by the sequences at the end + exponential amplification requires 2 primers
47
How do we amplify a segment bounded by a known sequence?
By using primers complementary to these ends in PCR
| Primers are designed to amplify a large target region
48
What is thermostability?
Ability to retain activity upon repeated heating to temperatures that would denature most enzymes
49
Why does the polymerase used in PCR have to be stable?
Because PCR involves multiple rounds of extreme heating + cooling
50
What is the PCR reaction based on?
Transition between 3 states
Denatured (template becomes single-stranded)
Annealed (formation of initiating template)
Native state at optimal extension temp. for enzyme activity
51
How many cycles of PCR are there usually?
30-40
Each cycle results in doubling amount of product, thus exponential accumulation
Reaction has characteristic kinetics determined by depletion of reactants + the acidification of the reaction
52
What is an SNP?
Single nucleotide polymorphism
53
How can we detect SNPs?
Methods depend upon difference in Tm conferred upon short sequences of DNA by their nucleotide composition
High resolution melting or probe based version of qPCR
54
What is high resolution melting?
Tm of amplified product is used to determine which sequence is present
55
What happens is probe based version of qPCR?
Sometimes called allelic discrimination
| Specific binding of probe to amplified region containing the SNP is detected
56
Forensics + law enforcement?
Amplification of genetic markers
Parentage of kinship
Identification (e.g. military casualties, missing persons)
Matching 2 sources (crime scene)
Authentication of biological matter (e.g. cell lines, purity of food)
57
What is an STR?
Short tandem repeat
2-5+ bases long, repeated many times at specific locations in genome
Highly polymorphic - number of repeats varies between individuals
58
How are STRs used in forensics?
Provide a pattern of uniquely-sized products accorded by each individuals genome providing a DNA fingerprint/molecular barcode
59
Primers + STRs?
Multiple sets of labelled primers are designed such that the products span different STRs
60
Other applications of PCR?
Amplifying material prior to NextGen sequencing + isolating individual segments of DNA prior to cloning sequencing
Manipulating + modifying DNA by introducing mutations into a DNA sequence or modifying the ends of the sequence to make them contain restriction sites/cloning vectors
Used in developing recombinant vaccines, pharmaceuticals (inteferons, clotting factors etc.)
61
Real-time/quantitative PCR?
End of PCR doesn't have a quantitative output + cannot be used to find out template copy number
Same end point as amplification becomes rate limited
So we use modified method called 'real-time PCR)
Utilise fluorescent detection of amplification
Used for quantifying amount of target DNA molecule in a sample
62
What do nucleases do?
Degrade nucleic acid by hydrolysing phosphodiester bonds
63
Examples of nucleases?
RNase degrades RNA
DNAse degrades DNA
Endonucleases degrade from one end of the molecule
Exonucleases cleave from within the nucleotide chain
64
What is a restriction endonuclease?
Restriction - limit transfer of nucleic acids from infecting phages into bacteria
Many different enzymes from different bacteria which recognise different specific DNA sequences
65
What do restriction endonucleases do?
Recognise specific sequence + cut it by catalysing the hydrolysis of phosphodiester bonds
66
Recognition sites/sequences?
4-8 bps in length, depending on the enzyme + palindromic
67
How often does a 4/6 base recognition sequence occur?
4 base = 4x4x4x4 = 256 bases
| 6 base = 4x4x4x4x4x4 = 4096 bases
68
What do nucleases produce?
Sometimes an overhang, sometimes blunt ends
69
What is a restriction map?
Map of restriction sites within a molecule
| Crude way of mapping unknown molecule + useful way to describe plasmids
70
What is DNA ligase?
Enzyme that repairs 'nicks' in phosphodiester backbone
| DNA molecules from different sources can be joined together to form a recombinant DNA molecule
71
What is DNA polymerase?
Enzyme that allows DNA synthesis in 5' to 3' direction
| Used in PCR, amplification, generation of probes, blunt-ending of DNA overhangs
72
What is phosphatase?
Enzyme that hydrolyses a phosphate group off its substrate
Calf intestinal alkaline phosphatase
Shrimp alkaline phosphatase
73
Why use a phosphatase?
To prevent cut plasmids from resealing
74
What is polynucleotide kinase?
Kinase = phosphate from ATP to substrate
| Polynucleotide kinase adds phosphate to 5' hydroxyl group of DNA/RNA
75
Why use a polynucleotide?
To phosphorylate chemically synthesised DNA so that it can be ligated to another fragment
To sensitively label DNA so that it can be traced using radioactively/fluorescently labelled ATP
76
What are probes?
Fragments of ssDNA
20-1000 bases in length
Complementary to gene of interest
77
What is reverse transcriptase?
RNA dependent DNA polymerase
Isolated from RNA-containing retroviruses
Synthesises a DNA molecule complementary to a mRNA template using dNTPs
78
Priming for reverse transcriptase?
Random primers -> cDNAs up to 700bp will cover length of all the RNA molecules
Oligo(dT) primers -> useful for cloning cDNAs + cDNA libraries, but some might not be full length
79
Where are plasmids found?
Many, but not all, bacteria
80
What are plasmids?
Discrete circular dsDNA molecules
Genetic elements (replicons) that exist + replicate independently of bacterial chromosomes
Therefore they are extra-chromosomal
81
Function of plasmids?
Means by which genetic info is maintained in bacteria
| Can normally be exchanged between bacteria within a restricted host range
82
What are vectors?
Cut down version of naturally occurring plasmids
83
What are vectors used for?
Used as molecular tools to manipulate
84
Factors of plasmid vectors?
Can be linearised at one or more sites in non essential stretches of DNA
Can have DNA inserted into them
Can be re-circulised without loss of ability to replicate at high multiplicity (copy number) within host cell
Contain selectable markers such as antibiotic resistance (e.g. ampicillin or tetracyclin)
Relatively small, often 4-5kb
85
How can we make + insert recombinant genes into plasmids?
Use PCR to amplify DNA, restriction enzymes to cut it + DNA ligases to rejoin it
86
How can we insert these plasmids into bacteria?
We can transduce bacteria where the plasmids will replicate + be maintained
87
Why would we want to isolate the plasmid which will express the recombinant gene?
To produce recombinant proteins in bacteria + investigate their properties/develop + produce therapeutics
88
Why use plasmids?
Plasmids add functionality over simple DNA
Expression of recombinant gene in prokaryotes + eukaryotes
Can add or modify control elements (e.g. inducible - switch on/off at will or express it to high levels on demand)
Alter properties of the gene product (e.g. make it secreted extra-celluarly/into periplasmic space or fuse it to a peptide tag or other protein)
Make it useful as a therapeutic
89
Recombinant proteins/peptides as biopharmaceuticals?
```
Make up 30%
Human insulin (diabetes)
Interferon-α (viral hepatitis)
Interferon-β (MS)
Erythropoietin (kidney disease, anaemia)
Factor XIII (haemophilia)
Tissue plasminogen activator, TPA (embolism, stroke)
```
90
What is meant by a constitutive promoter?
Always on
Allows a culture of cells to express the foreign protein at a high level
Fine if the protein isn't toxic to E.coli (bad idea if it is)
91
What is meant by an inducible promoter?
Molecular switch
Allows large cultures to be grown without expressing the foreign protein
Induced in response to a defined signal
Use transcriptional repressors
