Lecture 6: Molecular Techniques Flashcards
A good detection system should have what Three qualities?
Sensitivity, Specificity, and Simplicity
able to detect very small
amounts of target even in the presence of other molecules
Sensitivity
The test yields a positive result for the target
molecule only.
Specificity
he test must be able to run efficiently and inexpensively on a routine basis
Simplicity
What does CLIA stand for?
Clinical Laboratory Improvement Amendments
What does CMS stand for?
Centers for Medicare & Medicaid Services
What does CAP stand for?
College of American Pathology
What does JCAHO stand for?
the Joint Commission on Accreditation of Healthcare Organizations
What does COLA stand for?
the premier clinical laboratory education,
consultation, and accreditation organization
Degree of agreement between the nucleic acid sequences derived
from the assay and a reference sequence
Accuracy
Repeatability—degree to which the same sequence is derived in sequencing multiple reference samples, many times. Reproducibility—degree to which the same sequence is derived when sequencing is performed by multiple operators and by more than one instrument.
Precision
The likelihood that the assay will detect a sequence variation, if present, in the targeted genomic region
Analytical
Sensitivity
The probability that the assay will not detect a sequence variation, if
none are present, in the targeted genomic region
Analytical Specificity
The probability that the assay will not detect a clinically relevant sequence variation, if none are present, in the targeted genomic region.
Diagnostic Specificity
what are the steps for Isolating Nucleic Acids for
Molecular Analysis?
-lyze cells
-Enzymatic reactions to degrade proteins, lipids, other cellular molecules
-Chemical extraction and purification (organic and inorganic)
Isolating Nucleic Acids for
Molecular Analysis:
What is used for organic chemical extraction and purification?
Phenol-chloroform-isoamyl alcohol
Isolating Nucleic Acids for
Molecular Analysis:
What is used for inorganic chemical extraction and purification?
Salt precipitation, adsorption to silica surfaces/matrix columns), and anion-exchange chromatography
protocols.
Spectrophotometry of nucleic acid bases to determine concentration is measured at _____ absorbance.
260 nm***
OD 260 =
1.0 ~ 50 ug/ml of dsDNA or 40 ug/ml of RNA or ssDNA***
What absorbances indicates impurity?
Absorbance at 280 nm (protein absorption)
Absorbance at 270 nm (phenol contamination)
What OD suggests a pure sample?
OD260/280 ratio 1.8-2.0: pure preparation
Ratio < 1.8 contamination with proteins or phenol***
What kind of dye can be used for assessment of quality and quantity?
Fluorescent dyes
* with fluorometric or gel electrophoresis detection and quantification (EtBr, acridine orange, diaminobenzoic acid)
What methods are used for direct detection of NA?
-Restriction endonuclease enzyme digestion
-Gel electrophoresis and ETBr Stain
-Restriction Fragment Length Polymorphism (RFLP)
Restriction Endonucleases (RE) is only found in ___________.
microorganisms
Exhibit novel DNA sequence specificities
* >2000 distinct restriction enzymes have been identified
Restriction Endonucleases (RE)
Restriction Endonucleases (RE):
Function as ___________; recognize symmetrical dsDNA (palindromes)
homodimer
Utilized in the digestion of DNA molecules for hybridization procedures or in the direct identification of mutations
Restriction Endonucleases (RE)
- Palindrome reads the same in both directions
- Recognize specific sequences of _____________ nucleotides
4, 5, or 6
-Cutting genomic DNA with a RE results in many fragments of different sizes
restriction enzymes cut by breaking the __________ bond in both strands.
phosphodiester
-Sequences directly opposite one another on opposite strands of the dsDNA molecule
* Example: BamH1 (5’ overhang)
Gel electrophoresis separates molecules on the basis of their _______ and
______.
charge, size
NA Separation with Agarose Gel Electrophoresis:
molecules are sorted based on…
charge, size, and shape
What are the two types of gels used for gel electrophoresis?
-agarose
-acrylamide
Hb__ codes for normal b-
hemoglobin and produces
normal hemoglobin
Hb__ produces sickled red
blood cells.
A, S
Homozygotes for HbS are
anemic. (lost MstII site)
HbS produces b-globin that
differs from normal protein
by ____ amino acid
one
Types of hybridization techniques?
Southern, Northern, Western and Dot Blotting
Using specific probes that are labelled specific sequences of DNA can be identified.
Blotting/Hybridization Techniques
__________ Blot- Transfer of an RNA sample separated and identified
using DNA or RNA probes.
Northern
__________ Blot- Transfer of an DNA sample separated and identified
using DNA or RNA probes.
Southern
__________ Blot- Transfer of an Protein sample separated and
identified typically using an antibody.
Western
Probes
* ___DNA that will base pair with a complementary sequence of either RNA or DNA
ss
What reaction conditions affect annealing/hybridization?
Heat, chemical, Salt affect binding sensitivity and specificity
What can probes be labeled with?
- radioactive (P32) label
- chemiluminescent compound
- fluorescent compound
- enzymatic label (alkaline phosphatase/horseradish peroxidase with substrates to get color reaction done)
-Running Gel and transfer
Target DNA to membrane
-transfer of target DNA to membrane
-hybridization of DNA probe with membrane
-detection of complementary DNA bases
Sothern blot
Summary of southern blot
Separate DNA fragments by RE and gel electrophoresis. Hybridization with
Sequence specific probe
to analyze specific DNA sequences
- Allele specific probes can be made that are complementary
to a short strand of DNA that contains the most frequent
genetic mutations involved in a disease - Make normal and mutant probes
- Gene amplified by PCR followed by Dot-Blot (blotting technique)
Allele Specific Oligonucleotide Probes (ASO’s)
Cystic fibrosis - loss of phenylalanine ____ leads to one form of the disease
508
Allele Specific Oligonucleotide Probes (ASO’s):
ASO n =
ASO x =
WT
mutant
- Chemiluminescence detection of hybrid (DNA/RNA) molecules
- DNA is denatured
- Hybridized to RNA probe
- Captured by bound anti DNA/RNA antibodies
Hybrid Capture Assay
Capture Hybrids
* RNA:DNA hybrids are captured onto a
microtiter well coated with capture antibodies specific for RNA:DNA hybrids.
Label for Detection
* Captured RNA:DNA hybrids are detected with multiple antibodies conjugated to…
alkaline phosphatase
Permits billion-fold amplification of a selected region of a genome provided that at least a portion of its sequence is known
Polymerase Chain Reaction (PCR)
What are the steps for PCR?
- Denaturation- 94 C
- Annealing- temperature varies de[ends on the primer design to binds ssDNA template
- Extention (elongation)- 72 C
- thermostable Taq polymerase(DNA polymerase) from Thermophilus aquaticus
- Repeat for 2–30 cycles
- Terminattion- 40C?
- Detection of desired sequence
temp fo rTaq polymerase?
72 degrees Celsius (75-80)
Taq polymerase isolated from bacterium __________ that lives in hot springs and hydrothermal vents.
T. aquaticus***
Taq polymerase is able to withstand the protein-denaturing conditions (high
temperature) required during PCR and It replaced the DNA polymerase from _______ originally used in PCR
E. coli
Taq’s a half-life ?
greater than 2 hours at 92.5 °C, 40 minutes at 95 °C and 9 minutes at 97.5 °C
Taq pol can replicate a 1000 base pair strand of DNA in less than ___ seconds at 72 °C.
10
At 75-80 °C, Taq reaches its optimal polymerization rate of about ____ nucleotides per second per enzyme molecule
150
Examples of RT-PCR uses?
testing for HIV, or Covid
Reverse transcriptases are generally used to produce a DNA copy of the
RNA template using either random primers, an oligo(dT) primer or sequence-specific primers.
Rt-PCR
The ________ and ________ of the RNA template is crucial to the success of RT-PCR. Total RNA or poly(A)+ RNA can be used as the starting template, but both must be intact and free of contaminating genomic DNA.
* The efficiency of the first-strand synthesis reaction, which can be related to the RNA quality, also will significantly affect amplification results.
quality, purity
NASBA and TMA Isothermal PCR Begins with ______
RNA
What does RNAse H do?
separates RNA from DNA
What are the signal amplification methods?
- bDNA – Branched DNA probes
- Hybrid Capture – Anti-DNA-RNA hybrid antibody
Clinical usage of bDNA assays?
Versatile! methods have been developed for the detection
of infection by a wide range of microorganisms, including…
* parasite Trypanosoma brucei,
* cytomegalovirus,
* antibiotic-sensitive and antibiotic resistant Staphylococcus bacteria,
* human papillomavirus,
* hepatitis B virus.
More recent efforts have focused on the development of bDNA assays for the quantification of ___________ RNA, leading to the routine application of
bDNA methods in the clinical molecular diagnostics laboratory
HIV-1 and hepatitis C virus (HCV)
Two types of sequencing?
-Sanger sequencing
-Next Generation Sequencing
- Method to determine the exact order of the nucleotide
bases in DNA. - Unknown DNA sequences compared to known.
- Several methods available.
DNA Sequencing
What is the DNA sequencing method of choice?
Sanger
What dose Sanger method require?
ss DNA template, DNA primer, DNA polymerase, labeled nucleotides and
modified nucleotides to terminate DNA elongation
Sanger Method:
DNA sample divided into 4 separate reactions to normal (NTP) and ____ type of dideoxynucleotides (ddNTP) A, T, C or G are added.
ONE
Sanger Method:
_____ will prevent addition of further nucleotides
-Correct ratio of dNTP vs ddNTP creates DNA strands of discrete sizes
ddNTPs
Sanger Method:
- Each reaction loaded on ___________ lane on gel and electrophoresed.
- Sequence of nucleotides read in order to determine DNA sequence.
separate
Sanger Method:
to sequence, read the order of bases from the _______ to the ___________.
smallest, largest
fluorescence?-detecting laser, built into the machine, then shoots through the capillary fibre, causing the coloured tags on the DNA fragments, to fluoresce.
Each base terminator? is labelled with a different colour.
Chromatogram
What is the basic procedure of Next Generation of Sequencing (NGS)?
- Fragmentation of DNA
- Adapters are ligated
- Denature to single strands
- Form ation of clonal cluster or bead populations
Next Generation of Sequencing (NGS):
What are the major platforms?
- Ion torrent: powered by semiconductor chips technology
- Illumina (Solexa) sequencing based on sequencing-by-synthesis operations and reversible dye- terminators acquired from Manteia Predictive Medicine in 2004
The Purpose of High-Density Regions:
After Bridge PCR is conducted on the flow cell, Illumina uses fluorescently labeled ________ to detect each nucleotide bases.
So for example, if a red fluorescent light goes off, then we know it’s an A. If a blue light goes off, then we know it’s a G, and so forth (colors here aren’t accurate but you get the picture).
dNTP’s
What is the purpose of high-density regions?
the signal produced by the synthesis of one dNTP on a
strands is not enough to be detected. This is why we need to amplify the DNA sequences and producing a dense amount of sequences per area on the flow cell.
Illumina Sequencing-By-
Synthesis (SBS) Technology:
Clonal colony cluster creation
* the flow cells are subjected to isothermal bridge amplification, created clusters densities of up to ______ molecules. The duplication of each genomic strand aids in amplifying the generated signals upon sequencing.
2,000
Illumina Sequencing-By-
Synthesis (SBS) Technology procedure?
- Library Preparation
- Clonal colony cluster creation
- Sequencing- Bridge PCR/Sequence by synthesis
- Paired-end reads
- Output
Illumina Sequencing-By-
Synthesis (SBS) Technology:
Library preparation…
- tagmentation, addition of adaptor, sequence primer and index sequences
Illumina Sequencing-By-
Synthesis (SBS) Technology:
in order to elongate our reads, we may sequence starting from the other end. This would be helpful for….
de novo assemblies, detection of insertions/deletions, other
genomic mutations and solve ambiguous reads.
Bridge Amplification:
- Add _______ , and __________ enzyme to elongate DNA strands.
- Repeat until we have millions of dense clusters of DNA. The reverse strands are then cleaved and washed away.
dNTPs, DNA polymerase
Procedure of FISH Technique
- Harvest colcemid treated cultured cells in hypotonic solution
- Burst cell and load cells onto glass slide
- Protease and formaldehyde
treatment to clean cell debris - Denature the chromosomes
- Denature the probe
- Hybridization
- Fluorescence staining
- Examine slides or store in the dark
FISH was often used during M phase but is now used on ____ phase chromosomes as well.
I
Clinical utility of FISH?
- Identifies chromosomal abnormalities
- Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination
- Used to identify the presence and location of a region of DNA or RNA within
morphologically preserved chromosome preparations, fixed cells or tissue sections - This means you can view a segment or entire chromosome with your own
eyes
Use _____________-dUTP for FISH DNA Probe Labeling
Allylamine
-similar to northern or southern
-base-pairing, hybridization between nucleic acids
Microarray principle
Microarray principle:
Major differences from Northern?
-detects thousands of genes simultaneously/individual
-Probes fixation on glass slide
-Target samples labeling with fluorescent/radioactive dNTP
