Molecular Techniques Flashcards

Question

Describe and discuss Genome Mutation.

Answer 1

``` Affects the number of chromosomes Euploid - normal chromosome set Aneuploid – abnormal number of chromosomes Down’s Syndrome (Trisomy 21) Turner Syndrome (only one X chromosome) ```

Answer 2

Chromosomal abnormality in 95% of CML (Chronic myelogenous leukemia) T(9;22)(q34;q11) Gene on q34 referred to as Abl1 Gene on q11 referred to as BCR This translocation is a reciprocal exchange and is sometimes called the BCR/ABL fusion gene The protein complex formed results in the activation of the cell cycle and inhibits DNA repair

Answer 3

No it is just variation of genes in the population (Me).

Answer 4

Methicillin Resistant Staphylococcus Aureus mecA gene Encodes for penicillin binding protein 2a resulting in PBP2a which replaces the “normal” PBP1 Altered structure means penicillin can not bind properly, therefore unable to destroy the cell wall Two possible ways to acquire resistance Less than adequate dosages of antimicrobials builds resistance Passed to S. aureus by bugs that are intrinsically resistant

Answer 5

- Need samples in optimal condition - Ensure specimen integrity e. g. Is sample actually negative or is nucleic acid deteriorated due to poor sample collection, transport, storage, processing, etc. - Reduce or eliminate pre-analytical error by adhering to SOPs

Answer 6

Samples can include: whole blood, plasma/serum, urine, feces, buccal cells, spinal fluid, tissues, etc. Anything that contains nucleated cells!

Answer 7

1. PPE 2. Heparin contamination should be avoided 3. Use EDTA (lavender) or ACD (yellow) tubes. 4. Hemolysis should be avoided 5. Increased lipids can interfere with fluorescence detection methods 6. Use proper tubes for DNA collection, such as Polyallomer tubes 7. Storage requirements depends on nucleic acid being tested and method used. Refer to your facilities SOP

Answer 8

``` Protect the sample from Nuclease enzymes (degrade nucleic acids) found in your epidermal cells ```

Answer 9

EDTA Chelates Ca2+ and Mg2+ which helps to preserve cellular structure. Nuclease enzymes present in the sample require these as cofactors, so if they are consumed then there will be no nuclease activity

Answer 10

Not testing erythrocytes BUT, free hemoglobin interferes with enzyme activity in amplification techniques

Answer 11

Tubes Polypropylene and polyethylene tubes are known to adsorp DNA Polyallomer tubes are a specially designed polypropylene tube and have been shown to not adsorp DNA

Answer 12

Nucleic acid extraction: Release of nucleic acids from a cell for subsequent use. Should be free of any possible contaminants like salts, proteins, carbs, lipids, etc. Want to destroy cell not the nucleic acid.

Answer 13

Nucleic acid isolation: Isolating nucleic acid component from the rest of the cellular components so it can be analyzed

Answer 14

Some samples may require pre-treatment prior to extraction and isolation techniques: Centrifugation to get concentrated cellular component, grinding tissue to make more cells available for reactions, chemically treat bacteria/fungus to breakdown tough outer wall, etc.

Answer 15

1. High salt concentrations, proteases and alcohol 2. Cell lysis and protein degradation – dissolve lipids and proteins 3. Collect cellular debris 4. Precipitate with alcohol – isolate nucleic acids from cellular junk a) Precipitate can be collected and centrifuged b) Excess salt removed by rinsing in alcohol c) Re-suspended in buffer

Answer 16

Lyse cells Absorb DNA onto coated silica (column or magnetic beads) Positively charged silica beads attracts the negatively charged nucleic acid Everything else is waste DNA is eluted from the silica with buffer Elute is used for testing Goal: Nucleic acid with no contaminate See slide 32 for figure.

Answer 17

Be careful of RNase Degrades RNA RNA is less stable and more susceptible to damage Easily found Present in mammalian epidermal cells → sloughed off → degrade product = false negative Prevent RNase attack by:

Answer 18

Promptly processing samples, adding preservatives, using RNase free water/reagents/tubes/etc, and decontaminate regularly

Answer 19

Benefit of RNase? | Can be used to remove contaminating RNA if isolating DNA

Answer 20

Use UV Spec - DNA absorbs in UV spectrum, optimal at 260nm, due to purines and pyrimidines - Obeys Beer’s Law: Absorbance is directly related to concentration dsDNA concentration estimate (µg/mL) = A260 x dilution factor x 50 µg/mL ssDNA concentration estimate (µg/mL) = A260 x dilution factor x 40 µg/mL DNA Yield = DNA concentration x total sample volume Example: 600µg/mL = 1200µg/mL x 0.5mL

Answer 21

``` Contaminants also absorb in UV spectrum: Salt and ethanol = 230 nm Protein = 280 nm Use ratio: Ratio = A260/A280 1.6 – 2.0 = pure DNA 2.0 – 2.3 = pure RNA (higher due to the presence of uracil) 1.5 = equal amounts of protein and DNA <1.6 = Contaminated with proteins ```

Answer 22

1. Nucleases: Synthesize and/or degrade nucleic acids. Example: DNA polymerase and helicase 2. Exonucleases: Degrade from terminal ends of nucleic acids 3. Endonucleases: Degrade from within the sequence. a) Restriction endonucleases (aka restriction enzymes). Found in bacterium as an innate defense. Destroy bacteriophage; host DNA is methylated so no degradation.

Answer 23

1. Sticky ends: “Overhangs” 2-4 bp in length. 5’ G^AATTC 3’ 5’ - ----- 3’ 3’ CTTAA^G 5’ 3’ ----- - 5’ 2. Blunt ends: Both strands are terminated at the same bp. 5’ CAG^CTG 3’ 5’ --- --- 3’ 3’ GTC ^GAC 5’ 3’ --- --- 5’ Type II restriction enzymes recognize ~ 6 bp palindromes and cut sequence.

Answer 24

1. Cut large nucleic acids into smaller pieces to be analyzed by gel electrophoresis 2. Creation of recombinant nucleic acid molecules 3. Determine nucleic acid sequences. A restriction enzyme can be used to detect a mutation. E.g. if an enzyme recognizes & cuts a certain sequence of dNTPs that is considered normal you can test who is normal for that fragment. The non-normal ones will not have fragments cut and so will have larger bp on testing. See slide 41 for illustration of sickle cell anemia.

Answer 25

1. Can use restriction enzymes to create cuts (or not) in specific sequences a) Recognizes sequence = cut = smaller fragments b) Change in recognized sequence = no cut = larger fragments 2. Can infer that the polymorphisms a child possess is a combination of the parents. (Review examples on slide 43 and 44).

Answer 26

Polymorphisms are genetic variations that are inheritable.

Answer 27

1. Separation of molecular fragments (nucleic acids and proteins) in an electrical field. 2. Nucleic acids are separated based on their molecular weight. One nucleotide = one negative charge. Therefore, migration is inversely proportional to size. 3. Smaller nucleic acids (↓bp in length) migrate further and faster than larger nucleic acids (↑bp in length). 4. Smaller molecules migrate further and faster than larger molecules..

Answer 28

Basic operation: 1. Sample is added to wells in a neutral polymer medium that allows the passages of molecules when an electric current is applied. Examples: Agarose or Polyacrylamide. 2. Gel is submersed in a buffer which maintains pH and carries the electric current. 3. Negatively charged nucleic acids migrate towards the positive electrode.

Answer 29

1. Loading dye (aka tracking dye). Example: Bromophenol blue 2. Marks migration of nucleic acids 3. Added to sample prior to adding it to the gel. 4. Density agent (like glycerol – inert) to weigh sample down in well 5. Can tell if things are “right” within the first five minutes 6. Loading dye should run with the smallest fragment of nucleic acid Should migrate to ½ to ¾ the length of the gel.

Answer 30

Bands are made visible by staining with a gel: 1) Ethidium bromide (EtBr) is a potent mutagen that intercalates in the double helix. Excitation at 300nm causes EtBr to emit visible light at 590 nm (appears orange) 2. SYBR Geen is an alternative to EtBr. It sits in the minor groove of the double helix. It is more sensitive but more expensive, so not widely used.

Answer 31

Too ↑ = ↓ migration | Too ↓ = ↑ migration & susceptible to melting

Answer 32

Sample may denature | Incorrect migration

Answer 33

Too ↑ voltage = low resolution, heat could melt gel | Too ↓ voltage = minimal migration & sample diffusion

Answer 34

``` No amplification Degraded product Not stained Detection method not correct Gel misaligned (samples ran off) ```

Answer 35

Poorly made wells Overloaded samples Too high a voltage Degraded product

Answer 36

PAGE: Polyacrylamide Gel Electrophoresis, Smaller NA, separate to 1bp. PFGE: Pulsed Field Gel Electrophoresis, test conducted with alternating current directions, 24hrs+, good resolution of large fragments of nucleic acids (20,000-250,000 bps). Uses low agarose. Capillary: Narrow silica capillary tube, clear so fluorescence can be used. For nucleic acids of any size. Higher voltage used, heat dissipates easier from tube.

Answer 37

Identification of presence and location of specific sequences Need to create a hybrid Single stranded target sequence and single stranded probe with complementary base pairs anneal Conditions must be right: temperature, time, salt concentration, pH, presence of denaturants, etc. Referred to as stringency: ↑ stringency = lower chance of hybridization (only specific sequences) ↓ stringency = higher chance of hybridizations (similar sequences will match; more tolerance for mismatches) Allow probe and target to combine then detect!

Answer 38

1. Melting temperature, Tm: Temperature at which half of the nucleic acid is double stranded and half is single stranded. 2. Inflection point of melting curve: As dsDNA melts the absorbance increases. 3. GC pairs of stronger bonds = higher temps if found in higher concentrations 4. For short probes (14-20bp): Tm = 4ºC(#GC pairs) + 2ºC(#AT pairs)

Answer 39

Approximately 5degC lower than the probes melting temperature.

Answer 40

1. Only identify the target sequence 2. Avoid simple sequences and repeats 3. Will not self-anneal 4. Should be shorter as longer sequences may have reduced chances of binding (20-35 bases long) 5. Do not have a high GC content (will fold) 6. Use appropriate label for detection method 7. Radioactive versus non-radioactive

Answer 41

1. Databases like BLAST or GenBank provide public access to mapped sequences. 2. Ensure there are not similar sequences that may be present in your sample.

Answer 42

Southern Blotting (looking for DNA): 1. Use restriction enzymes – smaller fragments. 2. Electrophoresis to separate fragments. 3. Depurinate and denature gel - single stranded target. 4. Blot – solution drawn through gel transferring ssDNA from gel onto nitrocellulose paper or nylon (18+ hours) 5. Bake paper – permanently attach DNA to paper. (Alternatively use UV radiation to cause thymine to bind to amino acids in the membrane (referred to as UV crosslinking)) 6. Apply probe 7. Wash unbound probe 8. Detect

Answer 43

FRET (Fluorescence Resonance Energy Transfer): 1. Utilizes two specific probes: One with a 3’ fluorophore (acceptor) One with a 5’ catalyst for the fluorophore (donor) Example of donor acceptor pair = fluorescein-rhodamine 2. Within 1-5 bases of each other . . . Energy is transferred from the donor to the acceptor which causes the acceptor to lose energy by heat or fluorescence.

Answer 44

Amplicons Exponential amplification of a target sequence.

Answer 45

↑ yield by mimicking the in vivo nucleic acid replication process with the use of a master mix: 1. Deoxynucleotide triphosphate mixture (dNTPs) - dATP, dCTP, dGTP and dTTP that act as building blocks 2. DNase/RNase free water - diluent 3. Buffer with magnesium ions – maintain pH and provide enzymatic co-factor 4. Forward and reverse primers – flank target sequence and direct synthesis. Should have same characteristics as nucleic acid probe 5. Taq polymerase – synthesizes amplicons (extends primers by adding dNTPs). Enzyme is able to withstand high temperatures needed in the PCR cycle. 6. Nucleic acid template (extracted/isolated nucleic acid from patient sample)

Answer 46

Amplification occurs through a series of temperature changes. Use an instrument called a thermocycler An initial “hot start” at ~94˚C provides more single stranded target right at the beginning of the process, then continue into numerous PCR cycles (20-50): Denature – 90 to 96˚C – break H+ bonds so target sequence is single stranded Anneal – 50 to 70˚C – primers anneal Extend – 68 to 75˚C – primers are extended by polymerase enzyme to generate amplified product (amplicon)

Answer 47

PCR Product = 2^N (N = the number of cycles)

Answer 48

1. Positive Control - contains the target sequence, ensures reagents and instruments are working properly. 2. Contamination Control - no nucleic acids, ensures no contamination in system. 3. Negative Template Control - DNA without target sequence, ensures no mis-priming. 4. Amplification Control - another sequence is amplified, ensures master mix is working if patient result neg. Used to confirm negative result.

Answer 49

Amplicon identification: 1. Gel electrophoresis - Presence or absence of band, Location of band in comparison to molecular weight standard 2. DNA capture probes - Probes that are complementary to target sequence. Can use fluorescence labels.

Answer 50

1. Non-specific binding | 2. Use good primers & keep everything on ice.

Answer 51

Primers annealing to each other. | Confirm understanding

Answer 52

1. Presence of inhibitors (heparin, hemoglobin, etc.) 2. Degraded product 3. Not stained properly 4. Incorrect detection technique 5. Expired reagents or malfunctioning equipment

Answer 53

1. Amplicons can be aerosolized which is a major problem resulting in contaminated samples. 2. Prevent by: a) Decontaminate (bleach and 70% ethanol) b) One-way workflow (clean to dirty; never the other way Look at example of use of PCR controls on slide 68.

Answer 54

``` Reverse transcriptase PCR Two steps: RNA to cDNA Tht polymerase, a reverse transcriptase 45ºC-50ºC for 30 to 60minutes cDNA amplified Tht polymerase is deactivated at denaturing temp and Taq polymerase takes over amplification of cDNA Used to measure gene expression (BCR/ABL in Philadelphia Chromosome) or investigate organisms with RNA genome ```

Answer 55

Called real-time or quantitative PCR Compare sample curves to internal controls Product detected using probes as it is created Example: HIV viral load

Answer 56

Same target amplified by two subsequent reactions using two different primers for increased sensitivity and specificity

Answer 57

Multiple primers in master mix to look for multiple targets in one sample Must be careful with primer design – can’t anneal to each other or have different annealing temps! Example: Chlamydia and GC in urine or MRSA (identify bug and resistance)

Answer 58

Two primers come close to each other and DNA ligase joins them Amplify probes/primers or find point mutations

Answer 59

Data collected once every cycle Accumulation of fluorescent signal The Ct (cycle threshold) or Cq is defined as the number of cycles required for the fluorescent signal to cross the threshold Aka signal exceeds background level Ct levels are inversely proportional to the amount of target nucleic acid in the sample The lower the Ct the greater the amount of target nucleic acid in the sample Compared to the concentration of internal controls Refer to diagram on Slide 72.

Answer 60

Sanger Sequencing 1. Uses labelled ddNTPs – dideoxynucleotides ddATP, ddCTP, ddGTP, and ddTTP Lack 3’ hydroxyl group = polymerization stops as no phosphodiester bond can form 2. Creates fragments of various lengths that end with a labeled ddNTP 3. Still need primers and DNA polymerase to get various lengths (See slide 74 for illustration).

Answer 61

Pyrosequencing 1. Based on the release of pyrophosphate (PPi). In DNA synthesis we get the release of phosphate as each dNTP is incorporated and the phosphodiester bond forms. 2. The reaction will occur with one type of dNTP at a time 3. As DNA polymerase incorporates the specific dNTP PPi is released (creates a light signal). 4. Sulfurylase and adenosine 5’-phosphosulfate convert PPi to ATP 5. ATP converts luciferin to oxyluciferin = luminescent signal proportional to the amount of ATP. See illustration on Slide 77.

Molecular Techniques Flashcards

(85 cards)