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Molecular Techniques Flashcards

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1
Q

What is molecular biology?

A

Study of DNA, RNA, and proteins and their role in cell replication and the transmission of genetic information

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2
Q

What are molecular techniques?

A

Techniques implemented in order to study and/or manipulate DNA, RNA, or proteins
Examples:
- PCR
- Hybridization

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3
Q

Why perform molecular techniques?

A

Human Identification, identify organisms, determine genetic disease, molecular oncology, etc.

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4
Q

What is DNA?

A

Basic structure of life
Contains all of info to make you
Half from mom; half from dad
Housed in the nucleus in the form of chromosomes

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5
Q

Describe the basic organization of DNA.

A 
Organization:
Base pairs (bp) are the basic sequences 
Wrapped around proteins called histones
Histones make up nucleosomes
Several nucleosomes compose chromatin
Chromatin will make up a chromosome
Human genome = 23 pairs of human chromosomes comprised of 3.2 billion nucleotides
XX – female
XY - male
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6
Q

What is the smallest component of DNA?

A

Smallest component:
Deoxyribonucleotide triphosphates
Composed of a nitrogen base, a deoxyribose sugar and a triphosphate group

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7
Q

What are the four nitrogen bases that make up DNA?

A 
Pyrimidines – one ringed structures
- Thymine
- Cytosine
Purines – two ringed structures
- Adenine
- Guanine
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8
Q

By what kind of bond does dNTPs attach to one another?

A

dNTP’s attach to one another via phosphodiester bonds forming a string of nucleotides
Strong covalent bond between the 5’ carbon of one pentose sugar and the 3’ carbon of another pentose sugar
Attachment can only occur at the 3’ end
Attachment of nucleotide causes loss of two phosphates

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9
Q

What is the law of complementary base pairing?

A

DNA is double stranded
A purine and a pyrimidine form a base pair via hydrogen bonds
Cytosine – Guanine (3 hydrogen bonds)
Thymine – Adenine (2 hydrogen bonds)
Therefore, purines and pyrimidines will be in equal amounts
C-G form a stronger bond and require more energy to be separated.
Mismatching occurs when the laws of complementary base pairing are not met.

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10
Q

What is the result of the law of complementary base pairing?

A

Complementary base pairing creates a double helix
Sugar phosphate backbone (“sides of ladder”)
Phosphodiester bonds
Bases in the middle (“rungs”)
Hydrogen bonds
Protects unique base sequence
DNA is chemically stable

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11
Q

What are the chemical properties of DNA?

A

DNA is negatively charged, acidic, and hydrophilic
Bases are hydrophobic and protected in the interior
Polarity due to orientation

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12
Q

What determines DNAs polarity orientation?

A

5’ to 3’ direction from the phosphate terminal to the hydroxyl end
Strands are referred to as complementary
Relationship to one another is referred to as antiparallelism

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13
Q

What is RNA and its function?

A

RNA is the nucleic acid structure encoded by DNA
Function
To store and transmit “information” from the nucleus to the cytoplasm of the cell for protein synthesis

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14
Q

How is RNA different from DNA?

A
  • RNA possesses uracil instead of thymine
    Both are pyrimidine ring bases
    Thymine is methylated uracil
    Sometimes thymine is referred to as
    methyluracil
    Methylation is energy $
    Thymine in DNA prevents mutation
    Uracil is prone to forming bonds with other
    bases and deaminated cytosine looks like uracil
    RNA is single stranded but DNA is double stranded.
    RNA leaves nucleus but DNA does not.
    RNA’s sugar is ribose, DNA’s sugar is Deoxyribose
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15
Q

What are the different types of RNA?

A

Different types of RNA:

Pre-mRNA, mRNA, rRNA, tRNA, etc.

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16
Q

What happens in transcription?

A 
Transcription
DNA to RNA by RNA polymerase
Pre-mRNA to mRNA 
Introns removed by splicesomes only leaving exons
5’ guanine methylated cap
3’ poly A tail
Leaves the nucleus
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17
Q

What happens during translation?

A

Translation
RNA to protein
mRNA (codon for amino acid), tRNA (anticodon and amino acid), and rRNA
A and P site of ribosome > becomes amino acid chain with peptidyl transferase

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18
Q

What is reverse transcriptase?

A

Reverse transcriptase

RNA to cDNA

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19
Q

What is a mutation?

A

A mutation is an alteration in a gene or chromosome that causes diversity (positive or negative)
Affects the phenotype of the individual, and if it is a heritable change, can affect offspring
Phenotype – observable trait
Genotype - nucleotide sequence responsible for phenotype

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20
Q

Why is it difficult to determine which mutation caused a specific disease?

A

In summary there are so many mutations that occur it is hard to separate cause and affect. (Me)
From notes:
Hard to narrow it down to one specific mutation!
400 identified mutations that cause PKU by altering the PAH gene
600 identified mutations that cause Cystic Fibrosis
More than 2000 identified mutations that cause cancer susceptibility

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21
Q

What are some causes of mutations?

A

Some possible causes of mutations include:
Inheritance - mutations can be passed down to offspring
Error in DNA replication - spontaneous mutation caused by DNA not being repaired
Physical and chemical agents - mutagens affect DNA causing mutations

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22
Q

What is polymorphism? Name some examples discussed in class.

A

Polymorphism
Variation in the DNA sequence that is present in 1-2% in the population
Example: ABO Blood Groups
Example: Sickle Cell Anemia
Single base substitution → Glutamic acid (GAG) replaced by valine (GTG) = dysfunctional beta globin molecule in hemoglobin.
Con: Patient suffers from anemia
Pro: Malarial pathogens are unable to infect sickle cells

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23
Q

Describe and discuss gene mutations.

A

Affects a single or small group of genes
Types include:
Silent – nucleotide that does not change amino acid
Conservative – amino acid replaced by biochemically similar amino acid
Non-conservative – amino acid replaced by biochemically different amino acid
Non-sense – premature termination of translation
Frameshift – insertion or deletion of nucleotide that changes the whole message. Disasterous!

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24
Q

Describe and discuss chromosome mutations.

A

Affects structure of chromosome
Translocations – exchange of genetic information between chromosomes
Deletions - loss of chromosomal material
Insertions - gain of chromosomal material
Inversions - fragment of chromosome is removed, flipped, and reattached.

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25
Q

Describe and discuss Genome Mutation.

A 
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)
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26
Q

Describe the chromosomal abnormality found in chronic myelogenous leukemia.

A

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

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27
Q

Is polymorphism necessarily bad?

A

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

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28
Q

What mutation affected penicillin’s ability to fight Methicillin Resistant Staphylococcus Aureus
(MRSA)?

A

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

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29
Q

What pre-analytical considerations are required to assess DNA for clinical purposes?

A
  • 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
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30
Q

What specimens can be used to assess DNA for clinical purpose?

A

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

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31
Q

What practices are required to keep samples in optimal condition to ensure specimen integrity?

A
  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
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32
Q

Why is it important to wear PPE when handling DNA for clinical purposes besides protecting yourself from disease?

A 
Protect the sample from 
Nuclease enzymes (degrade nucleic acids) found in your epidermal cells
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33
Q

Why is it important to use EDTA (lavender) or ACD (yellow) tubes?

A

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

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34
Q

Why should hemolysis be avoided?

A

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

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35
Q

Why are polypropylene and polyethylene tubes not used and polyallomer tubes used instead for DNA sampling for clinical purposes?

A

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

36
Q

What is nucleic acid extraction?

A

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.

37
Q

What is nucleic acid isolation?

A

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

38
Q

What kind of pre-treatment techniques may be employed to extract/isolate nucleic acid from the cell?

A

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.

39
Q

Describe an overview of the basic steps in Nucleic Acid Extraction/Isolation.

A
  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
40
Q

Describe an overview of the basic steps in Nucleic Acid Solid Phase Extraction/Isolation using magnetic beads coated with silica.

A

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.

41
Q

What problems can RNase cause in RNA extraction/isolation?

A

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:

42
Q

How do you prevent problems with RNase in RNA extraction/isolation?

A

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

43
Q

When can RNase be of a benefit?

A

Benefit of RNase?

Can be used to remove contaminating RNA if isolating DNA

44
Q

How do you analysis of purity, and if extraction/isolation have no contaminants?

A

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

45
Q

How do you assess the purity of your DNA sample?

A 
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
46
Q

What are Nucleases, Exonuclease, and Endonucleases? What is a type of endonucleases, where is it found and what does it do?

A
  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.
47
Q

What are ‘sticky ends’ and ‘blunt ends’? What is used to create them?

A
  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.
48
Q

What is the application of restriction enzymes? Describe example of application.

A
  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.

49
Q

What is Restriction Fragment Length Polymorphism (RFLP)? Application example for paternity.

A
  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).
50
Q

What are polymorphisms?

A

Polymorphisms are genetic variations that are inheritable.

51
Q

What is electrophoresis?

A
  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..
52
Q

Describe the basic operation of electrophoresis?

A

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.
53
Q

What is used to help see how the DNA fragments are progressing in the gel?

A
  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.
54
Q

Loading dye helps just progress, but it does not help one to see bands. How are the bands made visible?

A

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.

55
Q

What issues can be caused with electrophoresis by too high or too low support in the gel medium?

A

Too ↑ = ↓ migration

Too ↓ = ↑ migration & susceptible to melting

56
Q

What issues can be caused with electrophoresis by an improper buffer?

A

Sample may denature

Incorrect migration

57
Q

What issues can be caused with electrophoresis by too low or high voltage?

A

Too ↑ voltage = low resolution, heat could melt gel

Too ↓ voltage = minimal migration & sample diffusion

58
Q

What issue can cause no bands in electrophoresis?

A 
No amplification
Degraded product
Not stained
Detection method not correct
Gel misaligned (samples ran off)
59
Q

What issue can cause smeared bands?

A

Poorly made wells
Overloaded samples
Too high a voltage
Degraded product

60
Q

Describe some other types of Electrophoresis Methods for Nucleic Acids (PAGE, PFGE, Capillary)?

A

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.

61
Q

Why are nucleic acid probes used?

A

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!

62
Q

What is melting temperature?

A
  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)
63
Q

What are typical annealing temperatures?

A

Approximately 5degC lower than the probes melting temperature.

64
Q

What are characteristics of a good probe?

A
  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
65
Q

What can help one identify a good target sequence for the probe?

A
  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.
66
Q

Describe the southern blotting technique for DNA?

A

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
67
Q

What is FRET (Fluorescence Resonance Energy Transfer)?

A

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.

68
Q

What are the amplified products referred to as in PCR? Purpose?

A

Amplicons

Exponential amplification of a target sequence.

69
Q

How do you get the exponential amplification of a target sequence?

A

↑ 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)
70
Q

What is the process of amplification?

A

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)

71
Q

How do you calculate the amount of PCR product?

A

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

72
Q

What controls are used in a PCR to ensure you can rely on your results?

A
  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.
73
Q

What is used for amplicon identification?

A

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.
74
Q

What is mis-priming and how do you avoid it?

A
  1. Non-specific binding

2. Use good primers & keep everything on ice.

75
Q

What is primer dimers?

A

Primers annealing to each other.

Confirm understanding

76
Q

What can result in false negatives in a PCR test?

A
  1. Presence of inhibitors (heparin, hemoglobin, etc.)
  2. Degraded product
  3. Not stained properly
  4. Incorrect detection technique
  5. Expired reagents or malfunctioning equipment
77
Q

What can cause false positives in a PCR test? How do you prevent?

A
  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.
78
Q

What is RT-PCR?

A 
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
79
Q

What is qPCR?

A

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

80
Q

What is nested PCR?

A

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

81
Q

What is Multiplex PCR?

A

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)

82
Q

What is ligase chain reaction?

A

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

83
Q

What is quantitative PCR?

A

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.

84
Q

What is sanger sequencing?

A

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).

85
Q

What is pyrosequencing?

A

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.

Clinical Chemistry 1 (11 decks)

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