Lecture 6: Molecular Techniques Flashcards by Sarah King

A good detection system should have what Three qualities?

Sensitivity, Specificity, and Simplicity

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able to detect very small
amounts of target even in the presence of other molecules

Sensitivity

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The test yields a positive result for the target
molecule only.

Specificity

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he test must be able to run efficiently and inexpensively on a routine basis

Simplicity

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What does CLIA stand for?

Clinical Laboratory Improvement Amendments

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What does CMS stand for?

Centers for Medicare & Medicaid Services

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What does CAP stand for?

College of American Pathology

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What does JCAHO stand for?

the Joint Commission on Accreditation of Healthcare Organizations

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What does COLA stand for?

the premier clinical laboratory education,
consultation, and accreditation organization

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Degree of agreement between the nucleic acid sequences derived
from the assay and a reference sequence

Accuracy

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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

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The likelihood that the assay will detect a sequence variation, if present, in the targeted genomic region

Analytical
Sensitivity

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The probability that the assay will not detect a sequence variation, if
none are present, in the targeted genomic region

Analytical Specificity

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The probability that the assay will not detect a clinically relevant sequence variation, if none are present, in the targeted genomic region.

Diagnostic Specificity

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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)

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Isolating Nucleic Acids for
Molecular Analysis:

What is used for organic chemical extraction and purification?

Phenol-chloroform-isoamyl alcohol

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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.

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Spectrophotometry of nucleic acid bases to determine concentration is measured at _____ absorbance.

260 nm***

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OD 260 =

1.0 ~ 50 ug/ml of dsDNA or 40 ug/ml of RNA or ssDNA***

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What absorbances indicates impurity?

Absorbance at 280 nm (protein absorption)
Absorbance at 270 nm (phenol contamination)

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What OD suggests a pure sample?

OD260/280 ratio 1.8-2.0: pure preparation

Ratio < 1.8 contamination with proteins or phenol***

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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)

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What methods are used for direct detection of NA?

-Restriction endonuclease enzyme digestion
-Gel electrophoresis and ETBr Stain
-Restriction Fragment Length Polymorphism (RFLP)

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Restriction Endonucleases (RE) is only found in ___________.

microorganisms

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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

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.

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.

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