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Semester 2 Revision > DNA Technology > Flashcards

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

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2
Q

Which nucleotides are pYrimidines?

A

CYtosine + thYmine (+ uracil)

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3
Q

Which nucleotides are purines?

A

Adenine + guanine

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4
Q

How many hydrogen bonds between cytosine and guanine?

A

3

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5
Q

How many hydrogen bonds between adenine and thymine?

A

2

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6
Q

How are sugar phosphates linked?

A

By phosphodiester bonds

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7
Q

Base stacking?

A

Hydrophobic interactions -> arrangement of bases set above each other internalised to the structure (+ excludes water)

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8
Q

VdW forces?

A

Individually small but contributes to stability

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9
Q

What does double-stranded DNA form?

A

Two antiparallel strands with negatively charged phosphates on the outside

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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

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11
Q

How is DNA denatured?

A

When DNA in solution is heated or using a strong alkali/urea

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12
Q

How can denaturing be measured?

A

Optically by absorbance at 260nm. Absorbance of light increases at 260nm.

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13
Q

What is hyperchromicity?

A

Increase of absorbance of a material

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14
Q

What is the melting temperature (or Tm)?

A

Point at which 50% of al strands separate

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15
Q

What is Tm dependent on?

A 
GC content
Length of DNA molecule
Salt conc.
pH (alkali is denaturant)
Mismatches (unmatched bps)
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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

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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

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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

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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
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20
Q

What is a mismatch?

A

A bp combination that is unable to form H-bonds

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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

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22
Q

What is the reversal of denaturation?

A

Renaturation

Formation of structure favours energy minimisation

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23
Q

What is renaturation facilitated by?

A

Cooling + neutralisation

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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)

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25
Q

Complementarity + Tm?

A

Basis of specificity
Perfect matches have higher Tm + are thermodynamically favoured over mismatches
Can use this property to form complementary molecules with no mismatches

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26
Q

Hybridisation + Northern blotting?

A

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)

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27
Q

What are other nucleic acid based techniques involving hybridisation?

A

Microarrays
Sequencing
PCR
Cloning

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28
Q

What is a probe?

A

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)

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29
Q

What is stringency?

A

Manipulating conditions - limiting hybridisation between imperfectly matched sequences increases specificity
High stringency - only complementary sequences hybridise
Temp near Tm
Low salt conc.

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30
Q

When are microarrays used?

A

To analyse expression of thousands of transcripts in each sample

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31
Q

Microarrays in Northern blotting?

A

Samples are fixed to solid surface (membrane) + a single probe is hybridised to a gene of interest

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32
Q

How are probes used in microarrays?

A

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’

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33
Q

What do ‘spots’ indicate in microarrays?

A

Lots of ‘spots’ = detection of lots of transcripts
Bright ‘spot’ = high expression level
Faint ‘spot’ = low expression level

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34
Q

What is gene expression profiling?

A

Simultaneously measure 50,000 different transcripts (in cell/tissue/organ)

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35
Q

Microarrays + SNPs?

A

Microarrays can assess millions of SNPs (up to 2.5mil simultaneously)
Used in association studies (e.g. gene association with disease)

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36
Q

What is PCR?

A

Enzyme-based method to specifically amplify DNA segments using thermal DNA polymerase in a cyclical process

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37
Q

How does DNA polymerise synthesise a new strand?

A

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
Q

How is the partially doubled-stranded structure formed?

A

By annealing a single-stranded primer to a denatured (thus single-stranded) template molecule

39
Q

What is annealing?

A

Essentially hybridisation of a primer

Results from formation of bps, stabilised by H-bonding between purines + pyrimidines

40
Q

Which is usually chemically labelled whilst the other one is not: a probe of a primer?

A

Probe is chemically labelled

41
Q

What are the primer reaction mixture concentrations?

A

Low copy number (low conc.) of template
Huge excess of primer which drives the reaction
Preferential annealing of the primer occurs

42
Q

What is DNA dependent DNA polymerase?

A

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
Q

What does DNA dependent DNA polymerase require?

A

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
Q

What is a chain reaction?

A

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
Q

Where does specificity stem from?

A

Primer complementarity

46
Q

Amplification of DNA segments?

A

Segment amplified is determined by the sequences at the end + exponential amplification requires 2 primers

47
Q

How do we amplify a segment bounded by a known sequence?

A

By using primers complementary to these ends in PCR

Primers are designed to amplify a large target region

48
Q

What is thermostability?

A

Ability to retain activity upon repeated heating to temperatures that would denature most enzymes

49
Q

Why does the polymerase used in PCR have to be stable?

A

Because PCR involves multiple rounds of extreme heating + cooling

50
Q

What is the PCR reaction based on?

A

Transition between 3 states
Denatured (template becomes single-stranded)
Annealed (formation of initiating template)
Native state at optimal extension temp. for enzyme activity

51
Q

How many cycles of PCR are there usually?

A

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
Q

What is an SNP?

A

Single nucleotide polymorphism

53
Q

How can we detect SNPs?

A

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
Q

What is high resolution melting?

A

Tm of amplified product is used to determine which sequence is present

55
Q

What happens is probe based version of qPCR?

A

Sometimes called allelic discrimination

Specific binding of probe to amplified region containing the SNP is detected

56
Q

Forensics + law enforcement?

A

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
Q

What is an STR?

A

Short tandem repeat
2-5+ bases long, repeated many times at specific locations in genome
Highly polymorphic - number of repeats varies between individuals

58
Q

How are STRs used in forensics?

A

Provide a pattern of uniquely-sized products accorded by each individuals genome providing a DNA fingerprint/molecular barcode

59
Q

Primers + STRs?

A

Multiple sets of labelled primers are designed such that the products span different STRs

60
Q

Other applications of PCR?

A

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
Q

Real-time/quantitative PCR?

A

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
Q

What do nucleases do?

A

Degrade nucleic acid by hydrolysing phosphodiester bonds

63
Q

Examples of nucleases?

A

RNase degrades RNA
DNAse degrades DNA
Endonucleases degrade from one end of the molecule
Exonucleases cleave from within the nucleotide chain

64
Q

What is a restriction endonuclease?

A

Restriction - limit transfer of nucleic acids from infecting phages into bacteria
Many different enzymes from different bacteria which recognise different specific DNA sequences

65
Q

What do restriction endonucleases do?

A

Recognise specific sequence + cut it by catalysing the hydrolysis of phosphodiester bonds

66
Q

Recognition sites/sequences?

A

4-8 bps in length, depending on the enzyme + palindromic

67
Q

How often does a 4/6 base recognition sequence occur?

A

4 base = 4x4x4x4 = 256 bases

6 base = 4x4x4x4x4x4 = 4096 bases

68
Q

What do nucleases produce?

A

Sometimes an overhang, sometimes blunt ends

69
Q

What is a restriction map?

A

Map of restriction sites within a molecule

Crude way of mapping unknown molecule + useful way to describe plasmids

70
Q

What is DNA ligase?

A

Enzyme that repairs ‘nicks’ in phosphodiester backbone

DNA molecules from different sources can be joined together to form a recombinant DNA molecule

71
Q

What is DNA polymerase?

A

Enzyme that allows DNA synthesis in 5’ to 3’ direction

Used in PCR, amplification, generation of probes, blunt-ending of DNA overhangs

72
Q

What is phosphatase?

A

Enzyme that hydrolyses a phosphate group off its substrate
Calf intestinal alkaline phosphatase
Shrimp alkaline phosphatase

73
Q

Why use a phosphatase?

A

To prevent cut plasmids from resealing

74
Q

What is polynucleotide kinase?

A

Kinase = phosphate from ATP to substrate

Polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA/RNA

75
Q

Why use a polynucleotide?

A

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
Q

What are probes?

A

Fragments of ssDNA
20-1000 bases in length
Complementary to gene of interest

77
Q

What is reverse transcriptase?

A

RNA dependent DNA polymerase
Isolated from RNA-containing retroviruses
Synthesises a DNA molecule complementary to a mRNA template using dNTPs

78
Q

Priming for reverse transcriptase?

A

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
Q

Where are plasmids found?

A

Many, but not all, bacteria

80
Q

What are plasmids?

A

Discrete circular dsDNA molecules
Genetic elements (replicons) that exist + replicate independently of bacterial chromosomes
Therefore they are extra-chromosomal

81
Q

Function of plasmids?

A

Means by which genetic info is maintained in bacteria

Can normally be exchanged between bacteria within a restricted host range

82
Q

What are vectors?

A

Cut down version of naturally occurring plasmids

83
Q

What are vectors used for?

A

Used as molecular tools to manipulate

84
Q

Factors of plasmid vectors?

A

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
Q

How can we make + insert recombinant genes into plasmids?

A

Use PCR to amplify DNA, restriction enzymes to cut it + DNA ligases to rejoin it

86
Q

How can we insert these plasmids into bacteria?

A

We can transduce bacteria where the plasmids will replicate + be maintained

87
Q

Why would we want to isolate the plasmid which will express the recombinant gene?

A

To produce recombinant proteins in bacteria + investigate their properties/develop + produce therapeutics

88
Q

Why use plasmids?

A

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
Q

Recombinant proteins/peptides as biopharmaceuticals?

A 
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
Q

What is meant by a constitutive promoter?

A

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
Q

What is meant by an inducible promoter?

A

Molecular switch
Allows large cultures to be grown without expressing the foreign protein
Induced in response to a defined signal
Use transcriptional repressors

Semester 2 Revision (3 decks)

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