Techniques 2 Flashcards
1
Q
What two processes entailed DNA extraction.
A
- Free max amount o fDNA from the cell.(DNA isolation)
- Separate DNA from other cellular material and debris. (DNA purification)
2
Q
Why do we do DNA extraction
A
- DNA located in nucleus enclosed in cell where there are other cellular components such as enzymes, proteins and organic and inorganic compounds. Therefore we need to separate the DNA from the contaminants.
3
Q
What are the basic steps of DNA extraction.
A
- Lysis and disruption
- Precipitation
- Purification
4
Q
Explain the lysis step of DNA extraction.
A
- The cell membrane and nuclear membrane need to be disrupted this can be either physical or chemical.
- Physical: mechanical disruption through homogenisation or sonication.
- Chemical: lysing using detergents (sodium-dodecyl-sulphate (SDS)) or enzymes (proteinase K,cellulase)
5
Q
How do detergents work to lyse cells.
A
Rapid disruption of membranes
6
Q
How do enzymes work to lyse cells.
A
Increase DNA recovery efficiency by degrading proteins and polysaccharides
7
Q
What does the precipitation step of DNA extraction entail.
A
Separate free DNA from cell debris and contaminants such as proteins, lipids, polysaccharides, detergents and reagents from lysis.
8
Q
Why is salt used in the precipitation step of extraction.
A
- Precipitates proteins -decreased solubility due to increased ionic strength of solution.
- Na+ neutralises DNA backbone and stabilises it
- With the addition of proteases, proteins precipitate out, DNA remains soluble in aqueous layer.
9
Q
Why is alcohol used in the precipitation step of extraction.
A
- Precipitates DNA -not soluble in alcohol
- Ethanol or isopropanol can bee used
10
Q
Explain what the purification step of extraction entails.
A
- Separate precipitated DNA from aqueous layer.
- Washing steps remove remaining debris and unwanted contaminants .
- Isolate pure DNA-dried and resuspended for storage
11
Q
Why do we do the salting out method
A
- High yield of DNA and eliminates most contaminates
- Cost effective
12
Q
Function of Tris-HCL (part of buffer )
A
Maintained pH
13
Q
Function of MgCl2 (part of lysis buffer)
A
Protects DNA from DNAse activity
14
Q
Function of triton-X
A
Detergent
15
Q
Function of sucrose (lysis buffer)
A
Osmotic membrane rupture
16
Q
Function of T20E5 (found in proteinase K solution)
A
- Maintains pH
- Chelating agent
- Inactivates DNAse
17
Q
Function of SDS (part of proteinase K)
A
- Lyses WBC lipid membranes
- Denatures protein
18
Q
Function of proteinase K
A
Degrades proteins
19
Q
Why should DNA be suspended in TE buffer
A
Stabilise DNA for longer storage
20
Q
Importance of DNA quantification
A
- Quantification of nucleic acids is done to determine the average concentrations of DNA present in a mix prior to downstream experiments.
- Sample purity is also an important consideration to accurately calculate the amount of DNA in a sample.
- The accurate measurement is based on sensitivity, specificity and interference by contaminants.
21
Q
DNA quantification methods
A
1.UV ABSORBANCE OR SPECTROPHOTOMETRY=Nanodrop
2.FLUOROMETRIC QUANTIFICATION=qubit fluorometer
3.AGAROSE GEL ELECTROPHORESIS=Gel tank + Agarose + DNA dye + Gel documentation system
4.AUTOMATED ELECTROPHORESIS + FLUORESCENCE MEASUREMENT= Bioanalyser or Tapestation
5.Real time PCR= qRT-PCR
22
Q
Explain spectrophotometry
A
- The spectrophometric method is based on the properties of a substance to absorb light at a certain wavelength
- .A spectrophotometer is the instrument used to illuminate the sample and measure the signal after the light passes through the sample
- The attenuation in the light that reaches the detector after passing through the sample is measured in relation to the incident light and expressed as absorbance values of the sample in the solution
4.The Nanodrop is an instrument employing spectrophotometry and is used for measuring the DNA quantity/ concentration and DNA purity/ quality of a DNA sample
23
Q
What do the 260/280 and 260/30 ratio mean
A
DNA is regarded as “pure” at:
-260/280 ~ 1.8 (pure RNA 260/280 ~ 2.0) and
-260/230 ~ 2
-Low ratios indicates contamination
-Residual contamination may result in an overestimation of the nucleic acid concentration
24
Q
Advantages of spectrophotometry
A
- Use small micro volumes (12microL)
- Rapid results for quick assessments
- Graph gives indication quality
- Concentration ranges from 2-15000, can be assessed.
- Quantification of concentrated samples avoiding the need for sample dilution
- Can provide information on contaminants -can identify non-nucleic acid contamination in samples
25
Disadvantages of spectrophotometry
1. Bad resolution for low concentration samples
2. Does not distinguish between DS or ssDNA
3. Containing samples leads to falsely high quantisation reading
26
How does a nanodrop work
1. Spectrophotometer passes light from a xenon flashlamp from the upper pedestal through the liquid and the lower pedestal where it is detected by the integrated spectrometer
2. Measure the absorbance at wavelengths of 230 nm, 260 nm and 280 nm
27
Advantages of fluorescence measurement
1. It is specific-performed measurement is selective for DNA, dsDNA, ssDNA and RNA.
2. It is sensitive -can measure pg/mL, it is the recommended method for very diluted nucleic acid samples.
3. It is accurate despite contamination being present in the sample, including nucleic acid contaminants
4. High throughput-microreader plate in one run.
28
Disadvantage of fluorescence measurement.
1. Time consuming - reagent and sample preparation are required.
2. No purity information is provided
3. Costly-expensive assay kit
4. Need specific equipment and reagent.
29
Advantages Agarose gel electrophoresis
1. DNA still usable afterwards
2. Requires a low amount of DNA
3. DNA can be detected, no matter the size
30
Disadvantages of agarose gel electrophoresis.
1. Time consuming
2. Low accuracy
31
Explain what The positive control for PCR is.
1. DNA with known outcome
2. Shows that the primers have attached to the DNA strand
32
Explain what The negative control for PCR is.
1. Sample without DNA
2. Shows if contamination of the PCR experiment with foreign DNA has occurred
33
Characterise primers used in PCR reactions.
1. Short oligo nucleotides: complement target sequence and bind to single stranded DNA.
2. 18-40 nucleotides long
3. Tm of 5C between each other
4. 50% GC content
5. Working solution (0.05microM-0.1microM)
34
Characterise dNTPs : Deoxynucleotide triphosphates
1. Nucleic acid components to generate new DNA.
2. 50microM each dNTP per reaction
35
What happens if there is excess dNTPs
Inhibits polymerase activity
36
What happens if the dNTPs concentration is to low.
Reduces amplicon yield
37
Function of DNA polymerase
1. It is a thermostable enzyme that synthesises copies of DNA with every cycle of PCR. It binds nucleotides to the complementary strand.
2. Binds first and add a nucleotide to the 3’-OH group of the primer . Moves in a 5’ to 3’ direction adding dNTPs.
38
Function of Mg2+/MgCl2 in PCR solution
1. Cofactors for DNA polymerase
2. Phosphodiester bond formation
3. Required for successful amplification
4. Concentration is higher than dNTP and primers.
39
Function of polymerase buffer in PCR solution
1. Facilitate amplification by stabilising the polymerase.
2. Most PCR buffer contain Tris/HCL at pH8
40
First stage of PCR -denaturation
1. Double stranded separate into two single strands.
2. Initial denaturing is needed before the three stages begin
3. Between 94-98C
4. 2min-10min: fully denature.
5. High GC content require higher and longer denaturation
41
Second stage of PCR-annealing
1. Primers anneal at complementary sequences
2. Ta-5C below lowest Tm of either primer.
3. Ta-52C-60C
4. Time -15-60seconds
42
What is the final stage of PCR : Extension
1. Temp allows optimal polymerase activity
2. Binding the nucleotides to the annealed primer.
3. 70C-80C -1-2minutes (this is polymerase specific.)
43
Visualising PCR product
1. Agarose gel electrophoresis
2. Capillary electrophoresis
44
Different types of PCR.
1. Hi fidelity PC
2. QPCR
3. Multiplex PCR
4. Hot-start PCR
45
Explain hi fidelity PCR
1. When down stream application require minimal errors
2. High-fidelity polymerase used for proofreading
3. Reduces number of replication errors
4. Cleaves out the errors and corrects mismatches
46
Explain multiplex PCR
1. Uses multiple pair of primers
2. Amplifies different target sequences
3. Within same DNA sample
4. Mostly kit based: not easy to optimise
47
Explain hot -start PCR
1. Reaction starts when specific temp is reached
2. Polymerase: inactive until required temp is reached
3. Reduces the nonspecific amp at low temps
48
How do you trouble shoot DNA templates that are low intensity bands or weak profile.
1. Quantity of input DNA and increase amount if necessary
2. Polymerase with high sensitivity for amplification
3. If appropriate, increase the number of PCR cycles
49
How do you trouble if there is something wrong with the primers.
1. Review primer design to make sure it’s Specific to target
2.check annealing temperature through gradient PCR
3. Optimise primer concentration
50
How to trouble shoot for suboptimal denaturing.
1. Check for the wrong temp
2. Low temps: No separation
3. High temps: Reduce enzyme activity
51
How to trouble shoot for suboptimal extension.
1. Amplicon length
2. Check DNA polymerase activity
52
How to trouble shoot for suboptimal annealing .
1. Do Gradient cycler
2. Check if Ta is 3-5°C below the lowest primer Tm
3. Consider polymerase used
53
Application of PCR in genetic disease.
1. Amplification of gene responsible for genetic disease makes it possible to detect mutation, position, size and nature
2. Possible to detect deletions, inversions, insertions, point mutations
54
diagnostic testing should be …
1. Affordable
2. Simple
3. Cost effective
4. Robust
5. Validation
55
Define analytical validity
Ability of molecular test to detect genetic variant
56
Define Clinical validity
Ability to correctly classify individuals to disease status or risk
57
Define clinical utility
Anticipated effects of clinical use of test results
58
What is Direct testing
Targeted testing for mutation associated with disease
59
What is indirect testing
1. comparison of DNA markers linked to trait of interest, they do not cause the genetic condition.
2. SNPs, STRs
60
How can PCR be USED FOR DIRECT MUTATION DETECTION in CYSTIC FIBROSIS (CF).
1. There are Many mutations (1000) for CF.
2. But there are Common mutations in different population groups – Blacks, Whites, Ashkenazi Jews
3. The Common mutation in Whites is ∆F508 (70-80%). The mutation is a Deletion of 3 bases in codon 508 of CFTR gene
4. PCR can be used to detect the 3 base deletion.
61
How can PCR be USED FOR DIRECT MUTATION detection in sickle cell anaemia
1. Sickle cell anaemia is caused by a Single base change (base substitution).
2. There is only 1 mutation in Central Africa (1/5 carrier), A-T substitution in codon 7 (GLU – VAL) in HBB gene
3. This can be Detect by using PCR and a Restrictions enzymes (REs)
4. The Mutation abolishes restriction enzyme site
4.1 No mutation – enzyme cuts
4.2 Mutation present- enzyme doesn’t cut
62
Detection of triple nuclei repeats using PCR
1. Can be Historically difficult to size TNR accurately because of Somatic variation or Interrupted repeats.
63
Why should primers be specific
1. Primers should ideally be complementary ONLY to the target
2. Most likely other regions exist with complementary or nearly complementary sequence – avoid homopolymer regions.
3. Non-specific products will only result from regions where: the two primers face each other on opposite strands and are a short enough distance away from each other.
64
Discuss primer length for qPCR
1. Affects melting temperature (Tm) Optimal 18-22 bases
2. Statistically, an 18-base sequence will be present only once in
every 418 bases (approx 69 billion, human genome = 3 billion bp)
3. Any shorter increases the potential for secondary annealing and non-specific product
4. Longer, does not improve specificity but increases melting temperature
65
What are melting temperature (Tm) and annealing temp (Ta)
1. Recommended: 55C to 70C
2. Differences in sequence, length, and composition of the primers, it is often difficult to have similar melting temperatures between the two.
3. Primers with melting temperatures in the range of 52- 58°C generally produce the best results
4. Ta Usually 5°C lower than the Tm
66
Why should GC-content be close to 50%.
Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.
67
Why Avoid complementary sequence between and within the primers…
To minimise primer-dimer formation
68
Possible causes of Low yield or no PCR product
1. Missing reaction component
2. Degraded DNA
3. Unsuitable or less optimal reaction conditions
4. Low primer concentration
5. Poor primer design
6. inhibitors present
7. Degraded nucleotides
69
How to remedy missing reaction component.
Check all components were added, set up new PCR.
70
How to remedy degraded DNA
Check DNA quality by gel electrophoresis
71
How to remedy unsuitable or less optimal reaction conditions
1. Decrease annealing T
2. Increase extension time
3. Optimise mg2+ concentration
4. Complete thawing and vortexing
72
How to remedy low primer concentration
Check primer concentration and increase if necessary.
73
How to remedy presence of inhibitors
1. DNA precipitation
2. Reduce amount of DNA used
74
How to remedy degraded nucleotides
Aliquot and minimise freeze/thaw cycles, keep on ice.
75
Possible causes for several non-specific products
1. Sub-optimal conditions
2. Poorly design primers
3. Primer concentration is too high
4. Contamination with another template
76
Remedies for sub-optimal conditions
1. Optimus Mg2+ concentration
2. Increase annealing T
3. Reduce elongated time and number of cycles
4. Set up quickly and on ice, add polymerase last.
77
Remedies for poorly designed primers
1. Check complementary especially at the 3’ end
2. Avoid >3 successive Gs or Cs at the 3’ end
78
Remedies for primer concentration is too high.
Check concentration and decrease if necessary
79
Remedies for contamination with another template
1. Separate pre- and post -PCR work spaces.
2. Wearer gloves, decontaminate work bench, autoclave tubes tips
80
What is real time PCR
1. Ability to monitor PCR reaction in real time
2. Accurate quantification of the amount of starting material (template) added to a reaction.
3. Reactions are characterized by the point in time during cycling when amplification of a target is first detected.
4. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
81
What are some uses of real time PCR.
1. Detection/and or quantification of pathogens in clinical samples
2. Allelic discrimination between wild-type and mutant or different splice forms
3. Virus sub-typing
4. Bacterial species identification
5. Measuring gene expression
6. Quantification of microRNA
7. Microarray validation
8. SNP genotyping
9. GMO testing
10. Measuring the effect of a drug
11. Gene knockdown from siRNA
12. Gene knockout
82
Basic principles of qPCR
1. PCR with a fluorescent dye that can be detected
2. PCR is theoretically exponential – product doubles with each cycle
3. The more starting material the faster the reaction will occur (lower cycle number)
83
Characterise Taqman assay
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.
84
Characterise the SYBR green dye
The SYBR Green dye chemistry uses SYBR Green dye, a highly specific, double- stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles.
85
Important difference TaqMan vs SYBR Green dye chemistries.
1. SYBR Green dye will detect all double-stranded DNA, including non-specific reaction products.
2. A well-optimized reaction is therefore essential for accurate results.
86
How to make Primers specific to experimental design.
1. Specific to target
2. 18 – 24bp
3. Produce product 50 – 150 bp in length (200bp does work)
4. Product ~50% GC and avoid GC stretches and repetitive sequences
5. Check for primer dimers especially for SYBR green assays
6. F and R primers annealing temp within 5 ̊C
87
Characteristics of SYBR green
1. Dye binds minor groove of all DNA
2. Fluorescence much stronger when bound
3. More product more dye bound
4. Cheap
5. Not specific
6.At early stage check product on gel and perform dissociation analysis (melt curve)
88
Characteristics of TaqMan
1. Dye and quencher either end of probe
2. Nuclease activity of polymerase removes dye and quencher during PCR
3. More product produced, more fluorescence
4. No melt curve
5. Allows multiplexing
89
Characteristics of simplex
1. One gene per PCR
2. SYBR green or Probe – more flexible
3. More variation
4. Usually simpler to set up
5. Uses more enzyme mix but can use SYBR green with cheap primers instead of expensive probes
6. More template
90
Characteristics of multiplex
1. Multiple genes per PCR
2. Probe based – 2 dyes (VIC FAM)
3. Minimises pipetting error
4. Competition between reactions can be problematic
5. Cheaper
6. Higher throughput – 2 measurements in one
7. Less template
91
What are the most common reference gene , and why are are they needed.
1. Most common: GAPDH, ACTB, UBC
2. Most common method of sample normalisation. They normalise a constant level of expression
92
Use of a no template control (NTC) during PCR.
(Should not see any amplification )
1. Detection of primer dimers
2. Contamination
93
The use of no reverse transcriptase control
1. Template contamination
94
The use of positive control
Checks that reagents and primers work
95
Use of a melting curve
By analyzing this curve, you can readily assess the homogeneity of the PCR products
96
Advantage of TaqMan assay
1. Specific hybridization between probe and target is required to generate fluorescent signal
2. Probes can be labeled with different, distinguishable reporter dyes, which allows amplification of two distinct sequences in one reaction tube
3. Post-PCR processing is eliminated, which reduces assay labour and material costs
97
Disadvantage s of TaqMan assay
Synthesis of different probes is required for different sequences
98
When troubleshooting real time PCR efficiency and amplification performance what should you check for.
1. RNA/DNA
2. Tissue degradation
3. Unspecific PCR products
4. Lab managment
5. DNA dyes
6. Cycle conditions
7. DNA concentration
8. PCR reaction components
9. Hardware:PCR platform
99
What is the first restriction enzyme isolated:
1. Hindill- haemophilus influenzae serotype ‘d’ found 2nd
100
How do bacteria protect themselves against their own RE.
1. Bacteria protect their self DNA from restriction digestion by methylation of its recognition site in its own genome
2. RE are methylation sensitive – they will not cut DNA if the recognition site is methylated.
3. There are some RE that will only cleave if the DNA is unmethylated.
101
Function of modification enzymes
1. The modification enzyme recognises the same sequence as the RE, but methylates DNA to protect ‘self’ against cleavage
2. Restriction enzyme and its cognate modification system constitute the R-M system
102
How are RE named
Genus: [E]scherichia
Species :[co]li
Strain: [R]Y13
Found: RE found [2nd]
Name= EcoRll
103
Recognition sequences vary between ………
Between 4-8 (can be longer)
104
Why are restriction enzymes with longer recognition sites preferred.
4bp^4 nucleotide bases=256 (on average cut once every 256 base pairs)
6bp^4nucleotide bases = 4096 (on average cut once every 4096 base pairs)
8bp^4nucleotide bases = 65536 (on average cut once every 65536 base pairs)
The longer the recognition site the less likely we are to get cleavage we don’t want.
105
What are isoschizomers
different enzymes that recognize and cleave in the same sequence. This means that researchers can choose between different RE that all have the same cleavage site to save money.
106
What is star activity. EcoRI*
Under non-optimal conditions RE’s can cleave similar sequences.
107
Contributing factors to non-optimal conditions
Contributing factors:
High enzyme:DNA ratios
High glycerol concentrations
High pH (>pH 8.0)
108
Characterise type 1 RE.
1. Cut at random, far from RE site
2. Little practical value because they don’t produce discrete banding patterns
109
Characterise type 2 RE
1. Cut at defined positions
2. Close to or within recognition site
3. Produce discrete restriction fragments & banding pattern
4. Require Mg2+
5. Used in labs for DNA analyses
110
Characterise type 3 RE
1. Cleave outside of recognition sequences
2. Exist as part of a complex with a modification methylase
111
Characterise type 4 RE
1. Cleave only modified DNA Like methylated DNA
2. Recognition sequences not well defined
112
Applications of RE.
1. Cloning : Gene fragment & plasmid DNA cut with the same restriction enzymes
Joined with a DNA ligase enzyme
2. Distinguish gene alleles :recognises SNP
113
Purpose of sequencing
1. Prediction of amino acid sequence
2. Identify genes that encode proteins
3. Detection of variation/changes: disease-causing or polymorphisms
4. Single nucleotide variants (SNV)
5. Small deletions, duplications and insertions
6. Used to assess genotype vs phenotype variations
7. Pathogenesis of diseases
8. Gene therapy
9.Personalized/precision medicine
10. Evolutionary relationships (species and populations)
114
Principle on how dideoxy nucleotides (ddNTPs)
1. Lack a 3’ hydroxyl on their deoxyribose required for the formation of a phosphodiester bond between two nucleotides
2. Incorporation of ddNTP prevents the addition of another nucleotide i.e. ddNTP cannot form a phosphodiester bond with 5’ phosphate group the next nucleotide
115
Limitations of Sanger sequencing
1.Targeted analysis (one target per reaction)
2. Short sequences 100 bp to 1000 bp (quality degrades after 700 bp)
3. Poor sequence in first 15 to 40 bases
4. Expensive and labour intensive for multiple targets (Works best for single gene disorders with know pathogenic variants)
116
Alternative to Sanger sequencing :next generation sequencing (NGS)
1. Higher sequencing depth enables higher sensitivity
2. Higher discovery power
3. Higher pathogenic variant resolution
4. More data produced with the same amount of input DNA
5. Higher sample throughput
117
Current applications of Sanger sequencing
1. Targeting smaller genomic regions (E.g. single genes)
2. Identifying known pathogenic variants (Familial variant +Common variants e.g. founder variants)
3. Validation of variants identified through Next Generation Sequencing (NGS)
4. Filling ‘gaps’ in NGS data in difficult-to-sequence areas and where coverage depth is low
(GC rich +Repetitive)
5. Verifying plasmid sequences and inserts
118
Process of Sanger sequencing
1. PCR amplification
2. Purification of PCR product
3. Cycle sequencing
4. Purification
5. Capillary electrophoresis
6. Analysis
119
Purpose of running a gel after a PCR reaction and before sequencing.
1. Determine if PCR amplification was successful
2. Determine if correct region was amplified
3. Determine if there was contamination
120
Why should PCR products be purified and what are the different methods.
Removes excess primers and dNTPs
Interfere with sequencing reaction
Different methods
1. Ethanol precipitation
2. Magnetic beads
3. Spin columns
4. Size exclusion membrane filters
5. Enzymatic
121
What are the components of cycle sequencing
1 .ddNTPs (fluoresently labeled)
2. DNTPS
3. Polymerase
4. Buffer
5. Primers (forward or reverse primer never both )
122
Why should excess/ unincorporated ddNTPs be removed from sequencing solution.
1. Interfere with quality and signal strength of sequencing data
2. Dye blobs – obscure portions of sequences
3. Base calling accuracy
