Lecture 6 Technique & Applications of Molecular Methods to Diagnosis Flashcards
What does sensitivity mean for a detection system?
Test must be able to detect very small amounts of the target even in the presence of other molecules
What does specificity mean for a detection system?
Test yields a positive result for the target molecule only
What does simplicity mean for a detection system?
Test must be able to run efficiently and inexpensively on a routine basis
CLIA
Clinical Laboratory Improvement Amendments
Regulate labs testing human specimens and ensure they provide accurate, reliable and timely patient test results no matter where the tests are done.
FDA
Food and Drug Administrations
Protecting public health by assuring the safety, effectiveness, quality and security of human and vet. Drugs, vaccines, and other biological products & medical devices.
CMS
Centers for Medicare & Medicaid Services
Oversees all lab testing, except some research, done on humans in the US through CLIA
CAP
College of American Pathology
Maintain accuracy of test results and ensure accurate patient diagnosis.
JCAHO
Joint Commission on Accreditation of Healthcare Organizations
objective of the Joint Commission is to continually improve and enhance the quality and safety of healthcare delivery in the United States. Towards this end, the Joint Commission makes a concerted, consistent effort to engage patients in issues associated with improving the quality and safety of healthcare delivery across the country
COLA
Premier clinical laboratory education, consultation, and accreditation organization
Improving healthcare & patient care in labs nationwide through accreditation. They are another form of certification.
Define accuracy in terms of genetic sequencing.
Degree of agreement between the nucleic acid sequences derived from the assay and a reference sequence
Define precision in terms of genetic detection systems.
Entails how well the system does in repeatability and reproducibility
Define repeatability in terms of detection systems
Degree to which the same sequence is derived sequencing multiple reference samples, many times.
Define reproducibility in terms of detection systems.
Degree to which the same sequence is derived when operating by multiple operators and by more than one instrument
What is analytical sensitivity?
Likelihood that the assay will detect a sequence variation, if present, in the targeted genomic region
What is analytical specificity?
The probability that the assay will not detect a sequence variation, if none are present, in the targeted genomic region.
What is diagnostic specificity?
The probability that the assay will not detect a clinically relevant sequence variation, if none are present, in the targeted genomic region.
What chemicals are used in lysing cells?
Detergent SDS, Tween 20, and etc.
What kind of enzymes are used in isolating nucleic acid for molecular analysis?
Enzymes that degrade proteins, lipids, and other cellular molecules.
What methods are used for chemical exaction and purification?
Two different kinds of methods. Organic or Inorganic methods.
What kinds of organic methods for extraction and purification are there?
Phenol-chloroform-isoamyl alcohol
What kind of inorganic methods are there for extraction and purification?
Salt precipitation
Adsorption to silica surfaces / matrix columns
Anion - exchange
Chromatography
Phenol-chloroform-isoamyl alcohol
An organic extraction. Used in precipitating cDNA after cells have been lysed at -20C or -80C (for an hour).
Salt precipitation method
Salt and ethanol are added to the aqueous solution, which forces the precipitation of nucleic acids out of the solution. After precipitation, the nucleic acids can then be separated from the rest of the solution by centrifugation. The pellet is then washed in cold 70% ethanol.
Adsorption to silica surfaces / matrix columns
method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions
Anion-exchange
salt concentration and pH conditions of the buffers used determine whether DNA is bound or eluted from the resin column
Chromatography
A column is first equilibrated with a solution containing DNA anion-exchange resin, which is used to selectively bind DNA with its positively charged diethylaminoethyl cellulose (DEAE) group. DNA is retained in the column while other cellular components such as proteins, lipids, carbohydrates, metabolites and RNA are eluted with medium-salt buffers. DNA can then be recovered by decreasing the pH or using high-salt buffers
How is spectrophotometry involved in assessment of quality and quantity of nucleic acid extraction?
It takes advantages absorbance of different molecules in the refined specimen.
Nucleic acids (want) absorb @ 260 nm
Protein absorption @ 280 nm
Phenol absorption @ 270 nm
Purification & Quality: OD260/280 ratio is over 1.8 means…
Pure preparation (good, I think)
Purification & Quality: OD260/280 ratio is under 1.8 means…
Contamination with proteins or phenol
What dyes are used in the detection and quantification of nucleic acid extraction?
EtBr, acridine orange, or Diaminobenzoic acid
Methods used for detection and quantification of genetic material?
Fluorometric or gel electrophoresis
Restriction Endonucleases function?
Function as a homodimer; recognize symmetrical dsDNA (palindrome).
What is the purpose of restriction endonucleases?
Utilized in digestion of DNA molecules for hybridization procedures or in the direct identification of mutations
Palindrome reads strands the…
Same in both directions (5’ and 3’)
Restriction enzymes recognizes specific sequences of…
4,5, or 6 nucleotides
How do restriction enzymes cut the strands?
Break the phosphodiester bond in both strands
Restriction enzymes cutting genomic DNA will result in…
Many fragments of different sizes.
The general rule with restriction enzymes. The smaller the recognition sequence….
The larger the number of fragments produced
Gel electrophoresis separates molecules based on their
Charge and size
How does the gel act in electrophoresis?
Gel acts as a sieve to slow the passage of molecules according to their size, shape, and charge.
How do particles move in gel electrophoresis?
Negatively charged particles move toward the negative electrode
Describe agarose composition
Complex polysaccharide derived from seaweed. Agarose gels are made of long chains of interlinked sugars to create a meshwork (like a spiderweb).
Describe polyacrylamide composition
Acrylamide is made by digesting acrylonitrile with nitrile hydratase. PA is made by the chemical crosslinking of acrylamide and bis-acrylamide. This linking produces a molecular sieve.
Which gel is used for a horizontal run?
Agarose
Which gel is used for a vertical run?
Polyacrylamide
Which gel type sets as it cools?
Agarose
Which gel type sets as soon as the chemical reaction once cross linking occurs?
Polyacrylamide
Use of agarose in electrophoresis?
Low resolution electrophoresis. DNA fragments of 50 - 20k bp in size. Resolution of over 6 Mb is possible with pulsed field gel electrophoresis
Use of polyacrylamide in electrophoresis?
High resolution. Gel has very high resolving power for small 5 - 500 bp fragments of DNA. Good for analyzing single stranded DNA.
Hb^A codes for
Normal Beta-hemoglobin and produces normal hemoglobin
Hb^S produces…
Sickled red blood cells.
Homozygotes of Hb^S are anemic and have lost the MstII site (one amino acid different).
In blotting / hybridization techniques use what to label specific sequences of DNA to identify them?
specific probes that are labeled specific sequences of DNA that can be identified.
What kind of material is examined in Northern blot?
RNA
What kind of material is examined in Southern blot?
DNA
What kind of material is examined in Western blot?
Protein
Which blots separate and identify using DNA or RNA probes?
Northern & Southern blot
What blot uses antibodies?
Western blot
In the hybridization method, probes will only hybridize with…
complementary sequence of either RNA or DNA
Probes are labeled with what in order to identify certain sequences in a strand?
Radioactive label (P^32)
Chemiluminescent compound
Fluorescent compound
Enzymatic label - Alkaline phosphatase & horseradish peroxidase combo w/ substrates to get color rxn.
ASO (allele specific oligonucleotide) probes can be made that are…
Complementary to a short strand of DNA that contains the most frequent genetic mutations involved in a disease.
What is followed after PCR when using ASO probes?
Dot-Blot a form of blotting technique.
In cystic fibrosis the loss of ______ leads to one form of the disease
Phenylalanine 508 leads to one form of the disease
Hybrid Capture Assay
Chemiluminescence detection of hybrid (DNA/RNA) molecules. The DNA is denatured then hybridized to RNA probe. The hybrid molecules are captured by bound anti - DNA/RNA antibodies.
After the capture of hybridized molecules how is it quantified?
It is detected with multiple antibodies conjugated to alkaline phosphatase. The concentration of the hybridized molecules is proportional to the intensity of chemiluminescent.
Describe Hybrid capture assay for HPV
Clinical specimen lysed with base solution to release target DNA. Hybridized RNA probe with target DNA to create a hybrid RNA/DNA product. I think its quantified with chemiluminescent … but not 100% sure.
PCR permits …
Billion fold amplification of a selected region of a genome provided that at least a portion of its sequence is known.
What are the steps of PCR?
Denaturation - 94C
Annealing - Temperature varies depends on the primer design to binds ssDNA template
Extension (elongation) - 72C
Thermostable Taq polymerase (DNA polymerase) from thermophilus aquaticus
Repeat for 2 – 30 cycles
Termination - 4C
Detection of desired sequence
Taq Polymerase is isolated from…
T. aquaticus living in hot springs and hydrothermal vents
Why is Taq polymerase used instead of other polymerases?
Its able to with stand protein - denaturing conditions required during PCR and it replaces DNA polymerase from E. coli originally used in PCR
Taq’s optimum temperature for activity is…
75 C to 80C, polymerization rate is about 150 nucleotides per second per enzyme molecule
Taq polymerase can replicate …
1000 bp strand of DNA in less than 10 seconds @ 72 C
What enzyme is used to produce a DNA copy from an RNA template using either random primers, an oligo (dT) primer or sequence - specific primers?
Reverse transcriptase
Once reverse transcriptase produces the cDNA template what happens after?
Basic PCR is carried out to amplify the target sequence
What is important to the success of reverse transcriptase PCR?
Quality and purity of RNA template
What enzymes are used in reverse transcriptase PCR?
Reverse transcriptase, primer 1, Primer 2, RNAse H, and Taq DNA polymerase
What methods are there for signal amplification?
bDNA - branched DNA probes
Hybrid Capture - Anti DNA-RNA hybrid antibody
Applications for signal amplification includes detecting what organisms?
Trypanosoma brucei
Cytomegalovirus
Antibiotic - Sensitive and antibiotic resistant Staphylococcus bacteria
HPV
Hepatitis B virus
What does DNA sequencing provide?
Method to determine exact order of nucleotide bases in DNA. Unknown DNA sequences compared to known. Sanger is the method of choice. Another method is NGS.
What is required to use the Sanger method?
Requires ssDNA template, DNA primer, DNA polymerase, labeled nucleotides and modified nucleotides to terminate DNA elongation.
What does ddNTPs in the Sanger method do?
Prevent addition of further nucleotides
In the Sanger Method, what is important in creating DNA strands of discrete sizes?
Correct ratio of dNTP versus ddNTP
Describe Sanger Method
DNA sample divided into 4 separate reactions to normal (NTP) and one type of dideoxynucleotides (ddNTP) A,T, C, or G are added. Each reaction loaded on separate lane on gel and electrophoresed
Sequence of nucleotides read in order to determine DNA sequence.
How do you read the DNA sequence in order after the Sanger method is complete?
To sequence, read the order of bases from the smallest to the largest. Diagram says the smallest is at the bottom of the electrophoresis gel. Largest is at the top close to the well (I think).
What is the basic major procedure in NGS?
Fragmentation of DNA
Adapters are ligated
Denatured to single strands
Formation of clonal cluster or bead populations
PCR amplifies DNA strands on flow cell or beads
NGS is an ultra or high ________ _________
throughput sequencing
illumina uses _________ dNTP’s to detect each nucleotide bases.
fluorescently labeled dNTP
What is done when the signal produced by the synthesis of one dNTP on a strand is not enough to be detected?
Amplify DNA sequences and produce a dense amount of sequences per area on the flow cell.
What goes into library prep?
Tagmentation, addition of adaptor, sequence primer and index sequence
Describe clonal colony cluster creation
Flow cells are subjected to isothermal bridge amplification, created clusters densities of up to 2000 molecules. The duplication of each genomic strand aids in amplifying the generated signals upon sequencing.
When are paired end reads useful?
For de novo assemblies, detection of insertion / deletions, other genomic mutations and solve ambiguous reads.
What is bridge amplification?
Add dNTPs, and DNA polymerase enzyme to elongate DNA strands. Repeat until we have millions of dense clusters of DNA. The reverse strands are then cleaved and washed away.
What is used for FISH DNA Probe Labeling?
Allylamine - dUTP
Describe FISH Technique
Harvest cells and place in hypotonic solution on glass slide to lyse cells.
Protease & formaldehyde to clean cell debris
Denature chromosomes
Denature probe
Hybridization
Fluorescence staining
Examine slides or store in dark
FISH Clinical Utilities?
-ID chromosomal abnormalities
-Aids in gene mapping, toxicological studies, analysis of chromosome structure aberrations, and ploidy determination
-Able to ID presence & location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections
- Often used during M phase but is now used on 1 phase chromosomes as well.
How is microarray similar to Northern / Southern blot?
Base-Pairing, hybridization between nucleic acids
What are the major differences from microarray to Northern Blot?
Detects thousands of genes simultaneously / individual
Probes fixation on glass slide / nylon membrane
Target samples labeling with fluorescent / radioactive dNTP
