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Year 2 Semester 1 > CMB2000 Strand A > Flashcards

CMB2000 Strand A Flashcards

1
Q

What is PCR

A

Polymerase Chain Reaction

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2
Q

What does DNA replication require?

A

-Template DNA
-40 or more proteins
-Helicase
-Primase
-Polymerases
-Nuclease
-Ligase
-ssDNA binding proteins
-Sliding clamps

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3
Q

What are the advantages of PCR?

A

-Sensitive (can amplify as little as one molecule)
-Specific (cam amplify a unique target sequence)
-Cheap
-Rapid
-Robust (DNA is very stable)

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4
Q

What’s required for PCR?

A

-Template dsDNA
-2 primers (small ssDNA)
-Polymerase (copies the template)
-dNTPs
-Magnesium (cofactor for DNA polymerase enzyme)
-Buffer (maintains pH and provide necessary salt)

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5
Q

What is the function of the sliding clamp in DNA replication?

A

The clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.

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6
Q

Describe the primers used in PCR?

A

-One for template and for complementary strand
-Single stranded DNA
-Length of 18-24 bp
-40-60%G/C content
-Start and end with G/C pairs
-Melting temp of 50-60C

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7
Q

What must primer pairs not have to prevent primer dimers?

A

Complementary regions to each other

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8
Q

Why is Magnesium required in PCR?

A

Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application

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9
Q

What makes up the buffer found in PCR?

A

-pH of 8-9.5
-Tris HCl
-Potassium ions (KCl) promotes annealing

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10
Q

What are the 3 stages of PCR?

A

-Denaturation
-Annealing
-Elongation

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11
Q

What is Nancy520 an example of?

A

An Intercalating agent, which aids in detection of DNA.

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12
Q

How may we detect PCR products?

A

Run products on agarose gel, using intercalating dye to stain DNA to determine size and yield.

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13
Q

Give some key steps involved in cloning

A

-Bioinformatics searching
-Design primers
-PCR
-Choose plasmid and insert
-Transform into competent E Coli
-Select correct colonies

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14
Q

Give some uses of PCR in biotechnology

A

-Cloning
-Manipulating DNA
-Knock out genes
-Fuse host proteins

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15
Q

What must our template DNA for PCR be?

A

-Clean and pure
-Contaminent free
-High concentration

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16
Q

Describe reverse transcriptase PCR

A

-Convert RNA to cDNA, using reverse transcriptase (a retroviral enzyme) that converts RNA to DNA
-Amplify DNA by PCR (including qPCR)

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17
Q

How does qPCR/Real time PCR work?

A

-Using a fluorescent report in the PCR reaction
-Couples amplification of the sequence with quantification of the concentration of DNA

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18
Q

How does qPCR using TAQman work?

A

Fluorescent that uses probes with fluorescent reporter and quencher

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19
Q

How does qPCR using SYBR green work?

A

Fluorescent that binds to groove of dsDNA

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20
Q

In qPCR, if there is a time to reach the cycle threshold, what does this signify?

A

more cDNA in the sample

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21
Q

What may we use as reference genes in qPCR?

A

-House keeping genes
-As these have a constant level of expression, which is not affected by experimental factors

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22
Q

Why do we use reference genes in qPCR?

A

-Essential to support validity of qPCR
-Confirms RNA extraction was good and efficient

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23
Q

Give some common uses of PCR in diagnosis.

A

-Genotyping the patient
-Genotyping the pathogen
-Phenotyping the disease

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24
Q

Why may we use PCR to genotype a patient?

A

-Diagnose genetic traits
-Detection of carriers of genetic traits
-Tissue matching (HLA typing)
-Pharmacogenetics

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25
Q

Why may we use PCR to genotype a pathogen?

A

Diagnose a strain of infecting pathogen

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26
Q

Why may we use PCR to phenotype a disease?

A

-Measuring disease progression
-Measuring disease severity

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27
Q

What types of PCR are used when genotyping a patient?

A

PCR-RFLP or ARMS-PCR

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28
Q

What is PCR-RFLP?

A

PCR-Restriction Fragment Length Polymorphism

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29
Q

What is ARMS-PCR?

A

Amplification Refractory Mutation System-PCR

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30
Q

How does PCR-RFLP work?

A

-Works when diseased alleles carry a site for a specific restriction endonuclease, but healthy does not
-When looking at bands, healthy and diseased can be differentiated

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31
Q

How may you identify diseased or healthy in PCR-RFLP banding?

A

-If healthy, there is only 1 band
-If homozygous for the diseased allele there are 2 bands
-If heterozygous with the diseased allele there are 3 bands

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32
Q

Give some disadvantages of PCR-RFLP

A

-Only possible if the site contains a known RE site
-Some RE are expensive
-Only possible if a single nucleotide variation
-Can be time consuming

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33
Q

Give some advantages of PCR-RFLP

A

-Cheap
-Easy design
-Applied to microindels and SNPs
-Simple resources with commonly used techniques

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34
Q

How does ARMS PCR work?

A

-One PCR comprises allele specific primer at 5’ end and a common primer at 3’ end
-If the presence of an amplified mutant is detected by agarose gel electrophoresis, it suggests that the target sequence contains the mutant allele

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35
Q

Give some advantages of using PCR over other methods of microbial diagnosis.

A

-Sensitive (can detect single copy of genome)
-Specific (Can identify strain)
-Quick, taking only a few hours
-No need for culture
-Detects DNA/RNA so not reliant on immune response

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36
Q

What may we use to phenotype a disease using PCR?

A

-Quantitative PCR
-Measures abundance of DNA or RNA in a sample

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37
Q

Why should we make competent cells instead of buying them?

A

-Saves money
-Ensures we don’t run out

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38
Q

What OD600nm should we grow competent cells to?

A

0.3, which is mid exponential growth

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39
Q

When forming competent cells, what may we plate them on?

A

Agar containing Xgal and ampicillin

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40
Q

Why do we use “double selection” ie plating competent cells on Amp+Xgal

A

-Ampicillin selects for bacteria that have taken up the plasmid (need resistance to survive)
-Xgal shows white colonies for the bacteria that have an insert as the lacZ gene no longer works and the lacZ makes the colonies blue

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41
Q

What is Xgal?

A

-When cleaved by βgalacotosidase creates an intensely blue product which is insoluble
-So when LacZ is functional blue product is formed.

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42
Q

Why do we use calcium chloride when forming competent cells?

A

-Disrupts cell membrane to allow plasmid uptake
-Aids binding of DNA to the surface of the cell, which can then enter the cell after a short heat shock

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43
Q

How can we ensure the proper orientation of the insert in PCR?

A

Using 2 different restriction enzymes

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44
Q

What is the percentage of agarose used in agarose gel electrophoresis?

A

1.5%

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45
Q

In Agarose gel electrophoresis, what is agarose dissolved in?

A

TAE

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46
Q

Name some reasons for DNA isolation

A

-PCR
-DNA analysis
-Genetic testing
-Forensics
-Ecological
-Archaeological

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47
Q

Name the steps of DNA isolation

A

1 Cell lysis
2 DNA purification from cell extract
3 DNA concentration
4 Measurement of DNA purity and concentration

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48
Q

What do we not want in a sample of DNA when isolating it?

A

-Proteins
-Ribosomes
-mtDNA
-Lipid
-Plasmids

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49
Q

Name some methods of cell lysis

A

-Biological methods
-Physical methods
-Mechanical methods

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50
Q

Describe biological methods to lyse cells

A

Using enzymes to disrupt cell membranes, different enzymes are required for different cells, eg
-For plants, cellulase is used
-For bacteria, lysozyme is used
-For eukaryotic cells, Sappanin is used

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51
Q

Give examples of physical methods to lyse cells

A

-Using osmotic pressure, ie excess water moves into the cell when cells are placed in a hypotonic solution, bursting it
-Using freeze-thaw, ie repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation

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52
Q

Describe mechanical methods to lyse cells

A

Grinding, ie
-Pestle and mortar is used to disrupt plant cells and hard tissue
-Bead mill is used to beat and grind tough samples
-Vortex is used with beads or by itself for small cell numbers

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53
Q

Describe DNA purification by phenol-chloroform extraction

A

-Lysed cells or tissue are mixed with equal volumes of a phenol-chloroform mixture
-These are then centrifuged forming two distinct phases as water does not mix with P-C mixture
-DNA is concentrated with 0.3M sodium acetate and 2.5 volumes ethanol, forming a DNA precipitate

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54
Q

Describe DNA purification using commercial kits

A

-Column contains a silica membrane that binds DNA in the presence of a high concentration of salt
-Impurities such as salts are washed away and then a low salt buffer is used to release the DNA from the membrane and collect it
-Results in purer DNA than phenol-chloroform extraction

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55
Q

Give some methods to measure quantity and quality of DNA

A

-UV absorbance
-Fluorescence dyes
-Agarose gel electrophoresis
-Capillary electrophoresis
-Diphenylamine

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56
Q

Why is DNA isolation and purification important?

A

-Efficient extraction leads to efficient science
-Without a good starting point there isn’t a good output
-Genomic testing wouldn’t be possible
-PCR/cloning wouldn’t work
-Forensic science would be unreliable

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57
Q

What are restriction endonucleases?

A

-Enzymes produced by bacteria to protect against viral DNA infection
-Act on specific DNA sequences (restriction sites)
-Cleaving the phosphodiester bond within a polynucleotide

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58
Q

How do restriction enzymes cut DNA?

A

-make one cut in each of the sugar phosphate backbones of the double helix (breaks bond between O and P) at their recognition site in the presence of Mg2+

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59
Q

What is Star activity of restriction endonuclease?

A

-Relaxation or alteration of the specificity
-When reaction conditions differ significantly from the optimum for the enzyme

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60
Q

Describe the principle of agarose gel electrophoresis

A

-Polymerised agarose is porous, allowing for the movement of DNA
-It acts as a molecular sieve
-Smaller, more charged molecules travel further
-Glycerol ensures DNA is heavy enough to stay in gel

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61
Q

What do we use to visualise agarose gel electrophoresis

A

Intercalating dyes

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62
Q

How is the size of DNA fragments in agarose gel electrophoresis determined?

A

-Size of product is compared to DNA ladder
-Look by eye
-Plot a Log10 graph and mm band migrated down the gel
-Using the MW ladder to create a standard curve

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63
Q

What is genome editing?

A

A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases -
-Enabling specific targeting of sequences within the genome without impacting the rest of the sequence
-Potential to cure genetic diseases in a patient specific manner

64
Q

What does CRISPR stand for?

A

Clustered Regulatory Interspaced Short Palindromic Repeats

65
Q

What is Cas9

A

CRISPR Associated Proteins

66
Q

What are the three components that make up the CRISPR complex?

A

-Cas9 (protein component)
-crRNA (RNA component)
-tracrRNA (RNA component)

67
Q

Describe how CRISPR acts as an adaptive immune regulator in prokaryotes?

A

-Invading DNA recognised is cut by Cat protein complexes into fragments termed protospacers
-Protospacers integrated into CRISPR locus located in the bacterial genome
-Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9
-Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
-Cas9 cuts DNA strand creating a double strand break to prevent reinfection

68
Q

Give some key components of the CRISPR locus

A

-Transactivating RNA
-Operon of cas genes encoding cas protein components (eg cas1, cas2, cas9)
-Identical repeat arrays
-Spacers of invading DNA

69
Q

Describe what guide RNA consists of in CRISPR

A

The complex formed between transactivating RNA and the protospacer/CRISPR RNA (crRNA) which enables selective binding of cas9 to invading DNA sequences

70
Q

What enables Cas9 mediated DNA cleavage?

A

Protospacer Adjacent Motifs (PAM), Cas9 will not cut invading DNA without a PAM site irrespective of Cas/gRNA binding

71
Q

How is CRISPR/Cas9 engineered for biomedical studies?

A

-The crRNA and tracrRNA is linked using a linker loop
-Deposition of the Cas9/gRNA complex at a desired locus of the genome will enable site-specific cleavage through nuclease activity
-The repair of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced

72
Q

What is required following CRISPR-mediated DNA cutting?

A

Cellular DNA repair pathways such as
-Homology directed repair (HDR)
-Non homologous end joining (NHEJ)

73
Q

Describe Non-homologous end joining (NHEJ)

A

Error prone as it
-Introduces insertions or deletions (indels) into DNA, generating frameshifts
-Impacts gene function
-Key to knockout studies

74
Q

Describe Homology Directed Repair (HDR)

A

-DNA is precisely repaired using sister chromatid during S phase (In CRISPR a template is introduced that will be used)
-PAM sites are removed from HR template to prevent retargeting of region
-Key to CRISPR knock in studies

75
Q

Describe Ex vivo methods to deliver CRISPR in the clinic

A

-Remove cells from the patient
-Edit genome
-Screen cell populations
-Engraft cells back into patient

76
Q

Describe In vivo methods to deliver CRISPR in the clinic

A

-Package CRISPR/Cas in a delivery vehicle
-Deliver to patient

77
Q

Describe how CRISPR has been used to knock out the Androgen receptor gene as a treatment for prostate cancer

A

1 Generate prostate cancer cell line expressing Cas9
2 AR gene locus was targeted by CRISPR to validate activity of Cas9, using NHEJ, introducing stop codon into AR gene
3 Prostate cell growth is reduced

78
Q

Describe how CRISPR has been used to knock in variants of the Androgen receptor gene as a treatment for prostate cancer

A

1 gRNA designed to exon 5 (ligand binding domain)
2 HDR template with point mutation encodes a stop codon, stopping formation of full length androgen receptor
3 Absolute loss of full length AR expression in CRISPR edited cells

79
Q

What should we consider with CRISPR cell therapies

A

-Efficacy of delivery
-Regulatory guidelines
-Mosaicism
-Specificity (off target effects)
-Immunogenicity
-Germline vs Somatic

80
Q

Describe how CRISPR is used to treat HIV?

A

-CCR5Δ32 is introduced into those with HIV
-This disrupts CCR5 coreceptor, which allows for a reconstitution of a functional immune system

81
Q

When did the Human genome project run?

A

From 1990 til 2003

82
Q

When was the first complete sequence of a bacterial completed?

A

1995, when H influenza was sequenced

83
Q

Why sequence the human genome?

A

-It is the blueprint of life
-Decipher what is coding and non coding
-Regulatory sequences
-Higher order structure
-Chromosome maintenance
-Comparative searches

84
Q

How is the entire human genome sequence obtained?

A

-Obtain the organisms genomic DNA
-Break the DNA into small fragments
-Search for overlaps to “reconstruct” the genome sequence

85
Q

Describe using model organisms in sequencing

A

-Small genome so “value for money”
-Easy organisms to manipulate
-Provide info on fundamental processes
-Useful in comparative genomics

86
Q

What were the outcomes of the human genome project?

A

-Provided a detailed map of the human genome
-Spurred the development of high throughput sequencing technologies, bioinformatics tools and data storage solutions, reducing cost
-Identified genetic variants associated with various diseases
-Fostered international collaboration among scientists

87
Q

What are some major issues of identifying genes within genomes?

A

-Identifying open reading frames
-Identification of RNA splice sites (as sequence changes when mRNA)

88
Q

Give some limitations of genome sequencing

A

-Incomplete coverage
-Does not capture the full extent of genetic variation
-Does not completely teach us function

89
Q

How has the human genome project aided research and medicine?

A

-Foundation for precision medicine (pharmacogenomics)
-Gene therapy
-Genetic screening
-Several open databases being accessible
-Comparative genomics (widely popular)

90
Q

Give the benefits of studies in model organisms

A

-Gives insight into functional characterisation of mutant proteins
-Understanding human genetic variation
-Crucial for developing personalised medicine
-Understanding the effects of mutation

91
Q

Describe using computational analyses of a genome to predict protein localisation

A

-Programmes predict the sub cellular location of a protein by statistically analysing patterns in its amino acid sequence
-eg PSORTII can determine if a protein is nuclear, cytoplasmic, cytoskeletal, vacuolar, etc

92
Q

What can computational analyses of sequence be used to do?

A

-Predict function
-Predict protein localisation
-Predict protein domains
-Identify regulatory sequences
-Characterisation of protein families

93
Q

What do functional genomics experiments describe?

A

Gene functions and interactions

94
Q

Give some types of functional genomics studies

A

-Protein/DNA interactions
-DNA methylation
-Gene expression
-Protein protein interactions
-Loss of function

95
Q

What do microarrays measure?

A

Hybridisation

96
Q

Describe different types of microarray experiments and what they sample?

A

Expression - cDNA
ChIP - Immunoprecipitation
SNP - Whole genome
Methylation - Whole genome
CGH - Whole genome

97
Q

Describe the method to microarrays measuring RNA expression

A

-Extract RNA and reverse transcript
-In vitro transcription with biotin labelled cRNA
-Fragment and hybridise with to GeneChip
-Wash away non specific binders and stain
-Scan array with lasers detect fluorescence with CCD and read image to computer

98
Q

Name what has replaced microarrays

A

High-throughput Sequencing

99
Q

Describe illumina sequencing

A

1 - DNA is fragmented and specific adapters are ligated to both ends of fragments
2 - This library is loaded onto a flow cell, where fragments undergo bridge amplification
3 - The flow cell is flooded with fluorescently labeled nucleotides, with each base having a different fluorophore
4 - Images are processed to identify the emitted fluorescence from each cluster, determining the sequence

100
Q

Describe RNA sequencing

A

-RNA is converted to cDNA
-cDNA used in high throughput sequencing technologies
-Used for sequencing library generation
-Allowing quantification, profiling and discovery of RNA

101
Q

Describe the steps of RNA sequencing

A

-Sample is PolyA enriched or rRNA depleted to focus on coding RNA, fragmented and adapters are ligated
-These fragments are reverse-transcribed and amplified
-These are then probed and read

102
Q

How is RNA PolyA enriched?

A

Done using a beat containing a sequence of Ts to which the Poly A tail will bind

103
Q

How is ribosomal RNA removed from a sample of RNA?

A

Done using a probe that bind to ribosomal RNA, and then dsRNAase enzymes are used to cut these

104
Q

What should we consider when using RNA sequencing?

A

-Big data sets require expert processing
-Expression data can be noisy
-Easy for confounding factors to dominate

105
Q

Describe ChIP sequencing

A

-Cells are treated with formaldehyde to crosslink proteins to DNA and preserve interactions
-Chromatin is fragmented
-DNA is enriched using immunoprecipitation
-Crosslinks are reversed through heating, and this DNA is sequenced
-Sequence data is analysed to identify binding sites of the protein across the genome

106
Q

Describe ATAC sequencing

A

-Nuclei are isolated
-Treated with transposes enzyme, inserting sequencing adapters into accessible chromatin
-DNA fragments are purified to remove unincorporated transposase
-Amplification
-Sequencing, and regions of open chromatin are identified, which correlate to regulatory elements like enhancers, promoters, etc

107
Q

Describe Bisulfite sequencing

A

-Genomic DNA is treated with sodium bisulphite, which converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged
-Bisulphite treated DNA is then amplified, where uracil is converted to thymine, resulting in a sequence where methylated cytosines are retained, while unmethylated appear as thymines
-These are then high throughput sequenced

108
Q

Describe reduced representation bisulphite sequencing

A

-Genomic DNA is first digested with restriction enzymes that cut near CpG, capturing regions of interest
-DNA fragments are size selected to enrich for fragments focusing on promoters and enhancer regions
-Selected fragments undergo bisulphite sequencing
-This allows for the study of key regulatory regions in epigenetic studies, allowing for a more cost effective way to study.

109
Q

Give the standard transgenic approach in mice?

A

-DNA is microinjected into the pronucleus of a fertilised mouse oocyte
-Injected oocytes are transferred to a pseudo-pregnant recipient mouse
-All offspring are screened for expression of the trans gene by DNA analysis

110
Q

Name some model organisms

A

-Mouse (Mus musculus)
-Clawed frog (Xenopus sp.)
-Zebrafish (Danio rerio)
-Fruit fly (Drosophila melanogaster)
-Nematode worm (C elegans)

111
Q

Give the gene targeted transgenic approach in mice?

A

-An isogenic trasngene with a drug selection gene is introduced into embryonic stem cells
-Drug selection is used and surviving cells are screened for the correct integration of the trans gene
-Correctly targeted cells are micro injected in mouse blastocysts
-Blastocysts are transferred to pseudo pregnant recipient mouse
-Chimaeric offspring are identified and mated to test for gremlin transmission of trans gene

112
Q

Give some strengths and weaknesses of transgenic mouse models

A

+Cheap and cheerful
+Multiple founders are generated
-Cannot control site of integration into genome
-Wild type gene product is still present and may interfere with phenotype

113
Q

Describe gene trapping (knockout mouse models)

A

-Gene trap vector is constructed containing a reporter gene and a selectable marker
-This is introduced into mouse embryonic stem cells using electroporation
-ES cells are selected using the selectable marker, only cells with the gene trap vector will survive
-Cells that survive selection are screened for the reporter gene
-Selected ES cells are injected into mouse blastocysts, which are then implanted into pseudopregnant female mice, producing chimeric mice
-These are then bred, to measure germline transmission

114
Q

Give some strengths and weaknesses of knock out mouse models

A

+Gene traps available for virtually all genes
-may not accurately model a known human disease
+Can make conditional knockout using cre recombinase
+Can stress or cross onto a different background

115
Q

What enzyme is used to insert a DNA fragment into a recipient plasmid?

A

DNA ligase

116
Q

What is the stage in cloning called in which we create copies of recombinant plasmids and stimulating cells to take up plasmids?

A

Transformation

117
Q

What is a multiple cloning site?

A

A short region of DNA containing several restriction enzyme recognition sites.

118
Q

What is the origin of replication

A

The DNA sequence that allows the initiation of replication. This is critical for DNA replication in the plasmid in host cells

119
Q

What is a selectable marker in cloning?

A

A gene encoding a product which protects the organism from a selective agent that would normally kill it; this allows you to select for bacteria which have taken up the plasmid. An antibiotic resistance gene is commonly used.

120
Q

Give the order found in plasmids expressing a protein of interest?

A

-Promotor
-Ribosome Binding site
-Start codon
-Affinity tag
-Stop codon
-Transcription terminator

121
Q

What is the role of an affinity tag?

A

The affinity tag encodes a small peptide that will be incorporated into the translated protein. It facilitates the purification of the expressed protein. The MCS contains several restriction enzyme sites, allowing the gene of interest to be inserted.

122
Q

What is used to spread bacteria on an agar plate?

A

L spreader

123
Q

During clonal selection, what does it mean if only the positive control plate has growth, but the sample and negative control have no growth?

A

Issue with digestion or ligation of plasmid

124
Q

In blue-white screening theory, which colour colony do we want?

A

The white colonies represent the most successful cloning results. The bacteria in the white colonies have been successfully transformed and these bacteria contain not only the plasmid of interest but also the desired insert.

125
Q

What is the function of lac repressor protein

A

The repressor protein prevents LacZ transcription occuring constantly, which would unnecessarily waste resources in the cell.

126
Q

To remove the lac repressor, a reagent called IPTG can be added.
This binds to the lac repressor, changing its shape and causing it to dissociate.
Predict the effect of adding IPTG to β-gal expression?

A

When IPTG is added, the repressor lifts off the operator. This allows more RNA polymerase to bind, increasing the transcription (and later translation) of LacZ into β-gal.
Therefore IPTG can be seen as an activator of the lac operon and β-gal expression.

127
Q

What are minipreps?

A

Protocols designed to isolate and purify plasmid DNA from a bacterial cell culture

128
Q

Give the steps in miniprep (isolating plasmid DNA)

A

-Centrifugation
-Resuspend pellet (remove growth medium)
-Cell lysis
-Neutralise pH
-Discard Pellet

129
Q

When lysing cells (during DNA isolation), how should we mix alkaline lysis buffer?

A

The tube should be inverted gently to ensure adequate mixing, vortexing could shear chromosomal DNA (making it harder to separate from the plasmid)

130
Q

What does neutralisation buffer contain when isolating plasmid DNA?

A

-Weak acid and chaotropic salt
-Used to decrease alkalinity from lysis buffer

131
Q

What is the function of chaotropic salts in neutralisation buffer (used in miniprep isolating Plasmid DNA)?

A

-Disrupt hydrogen bonds, keeping larger DNA and proteins denatured
-Genomic DNA is too large to renature and aggregates with the SDS, protein and cellular debris which can be easily removed

132
Q

Describe the steps in purifying plasmid DNA

A

-Bind DNA: Pour supernatant into a miniprep column. The plasmid DNA binds to the silica.
-Wash: Add high salt buffer to remove impurities such as cell membrane debris.
-Plasmid elution: Add low salt buffer to release plasmids from membrane ready for collection.

133
Q

What is the most suitable buffer to wash the silica membrane when purifying plasmid DNA?

A

Passing high salt buffer through the column will help wash off a range of impurities, such as fragments of cell wall or cell membrane.
The high salt concentration ensures that the plasmid DNA remains tightly bound.

134
Q

Once the collection tube is discarded, and the purified plasmid DNA is bound to the silica membrane, which buffer should be used to elute the plasmid DNA?

A

A low salt buffer will disrupt the bonds between the plasmid DNA and silica membrane so can be used to elute the DNA from the column.

135
Q

What do positive and negative controls in PCR contain?

A

-Positive contains DNA known to contain region X
-Negative contains DNA with no region X

136
Q

What is the combined effect of SDS and a reducing agent in SDS PAGE?

A

-Breaks up disulfide bridges
-Keeps the protein denatured during electrophoresis.
-Ensures that SDS-PAGE separates proteins on the basis of size only, rather than needing to account for size and native charge.

137
Q

What are the loading dyes?

A

Dyes that allow you to visually track migration of the sample through the gel

138
Q

What are the functions of APS and TEMED in SDS PAGE?

A

APS and TEMED initiate polyacrylamide formation, and bisacrylamide is needed for cross-linkages and gel integrity.

139
Q

Describe the Molecular Weight standard curve in SDS PAGE analysis?

A

The log(MW) is plotted against the relative migration distance along the gel for each of the MW standards. There is a linear relationship between the two variables.

140
Q

What goes on the X axis and the Y axis on a molecular weight standard curve in SDS PAGE?

A

X axis - Relative migration distance
Y axis - Log(MW)

141
Q

How do you calculate the molecular weight in kilodaltons from Log(MW)

A

Molecular weight = 10^logMW

142
Q

Give the steps in SDS PAGE staining

A

-Staining the gel and proteins
-Destaining solution is added to remove the background stain
-Multiple rounds of destaining are usually required to remove the background stain

143
Q

Describe the steps in western blotting

A

SDS PAGE - The proteins have been separated according to their molecular weight
Transfer - The proteins are eluted from the gel onto a porous membrane. An electric field is used to transfer the proteins to the membrane.
Immunoblotting - The membrane is probed with primary antibodies specific to the protein of interest. Secondary antibodies bound to an enzyme bind the primary antibodies
Detection - Enzyme substrate is added. If the protein of interest bound by enzyme-conjugated antibody, the enzyme will catalyse. This will produce a detectable chemiluminescent (light) signal where protein is bound.

144
Q

Describe the function of the transfer cassette in a “blotting sandwich” in western blotting?

A

The cassette holds the sandwich components together. After assembling the blotting sandwich, the cassette is closed and placed in the transfer tank.

145
Q

Describe the function of the membrane in a “blotting sandwich” in western blotting?

A

A porous membrane made of either nitrocellulose of PVDF, which has a high affinity for binding proteins. Proteins move from the gel to the membrane

146
Q

Where should the membrane be positioned in a “blotting sandwich” in western blotting?

A

Between the gel and the anode so it catches the proteins as they move towards the positive electrode.

147
Q

What is the role of a rocker in western blotting?

A

Ensures the reagents are distributed evenly across the membrane

148
Q

What is the purpose of blocking solution during transfer in western blotting?

A

To prevent the binding of antibodies to areas of the membrane not occupied by sample protein, by binding to areas of the blot not occupied by sample protein.

149
Q

Give some common blocking solutions used in western blotting

A

-Non-fat dried milk
-BSA

both diluted in Tris buffered saline mixed with tween 20

150
Q

When washing unbound primary antibody, Tween 20 (non ionic detergent) is used. Why?

A

To help reduce the non specific binding of antibodies in subsequent steps

151
Q

Describe what secondary antibodies must be in western blotting?

A

Must be specific for the host species in which the primary antibody was generated but must not have been raised in that species itself.

152
Q

Give some secondary antibody-bound enzymes typically used in western blotting

A

Horseradish Peroxidase (HRP) and Alkaline phosphotase (ALP)

153
Q

What is the function of a loading control in western blotting?

A

-Confirms that the same amount of total protein has been loaded into each lane of the gel.
-Provides a baseline for comparing the expression levels of the protein of interest across samples.
-Verifies that observed differences in target protein levels are due to biological changes, not technical errors.

154
Q

Give the order in which ELISA should be carried out

A

-Capture antibody
-Blocking buffer
-Sample
-Primary antibody
-Secondary antibody
-Enzyme substrate

155
Q

In qPCR, what is the cycle threshold?

A

The cycle threshold (Ct) is the number of cycles required for the fluorescence signal of the target DNA (or cDNA) to cross the threshold level, which is set above the background fluorescence.

156
Q

What does cycle threshold signify?

A

Low Ct: High initial target concentration.
High Ct: Low initial target concentration.

Year 2 Semester 1 (5 decks)

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