Module 6: Detection and Analysis of Nucleic Acid Flashcards
Macromolecules that exists as polymers
called polynucleotides.
Nucleic acid
Formation of a nucleotide by adding ____ group to a nucleoside
phosphate groups
phenol and chloroform/isoamyl alcohol
(25:24:1) are mixed with an equal
volume of samples by vortexing
Organic purification
involves the incubation of nuclei with only proteinase K at 65°C
Inorganic Purification
movement of DNA or RNA in response to an electric field
Electrophoresis Separation
DNA are reduced to fragments by digestion with ____.
restriction enzymes
are small molecules and no digestion is required prior to electrophoresis.
RNA
Routinely uses Gel electrophoresis.
Uses TAE or TBE.
Molecular separation of DNA
interaction between single stranded nucleic acid to form a (duplex) double stranded molecules based on complementary base
pairing of the sequences.
Nucleic Acid Hybridization
process of recombination of two non-labeled strands into a stable double stranded structure.
annealing
If one strand has a marker, a hybrid is formed between labeled and an unlabeled strand.
hybridization
labeled strand is called the ____ and the product is called ____
probe; hybrid
sample and probe in a solution
Liquid or Solution Phase Hybridization
e.g. Digene Hybrid-Capture 2, Digene Corp. USA
Liquid/Solution Phase Hybridization
Hybridization assay formats for detection of HPV
Liquid or Solution Phase
occurs in biphasic environment
Solid Support Hybridization
solid phase in solid support hybridization
sample
liquid phase in solid support hybridization
probe
e.g. dot or blot
Solid Support Hybridization
What are the two Hybridization assay formats
Liquid or Solution Phase Hybridization
Solid Support Hybridization
described by E.M. Southern in 1975, detect specific DNA sequences and gene mutations
Southern Blot
Blotting techniques in which examples are electrophoretic separation, transferring to solid support nitrocellulose and hybridization
Southern blot
Blotting technique that uses RNA sample
Northern Blot
RNA is extacted, digested, electrophoresed, blotted and probed.
not routinely used in clin lab because RNA is not stable
Northern blot
in northern blot technique, how many minutes and in what temp is rna denatured?
15 min of 55 C heating
used to identify proteins
separated by PAGE
Western Blot
confirmatory for HIV and used in the diagnosis of Lyme Disease
Western blot
membranes used in western blotting
- nitrocellulose
- nylon
- PVDF or Polyvinylidene difluoride
probe molecule in southern and northern blotting
DNA
western blotting probe molecule
antibody
south-western blotting probe molecule
dsDNA
Results from a variable number of tandem repeats (VNTR) in a short DNA FRAGMENT.
used in FORENSIC DIAGNOSIS, DONOR TRANSPLANTS.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
In RFLP, restriction fragments are separated on gel and analyzed by ____ blotting techniques.
southern
*amplified nucleic acids, whether an RNA or DNA are referred to as
amplicons
patented the PCR method in 1985
Kary Mullis
target amplification method if the nucleic acid of interest is RNA rather than DNA, conversion of RNA to DNA
reverse-transcriptase PCR
TA method which uses numerous primers for different targets in the same mixture
Multiplex PCR
target amplificationa nd detection steps occur simultaneously and PCR product is detected using fluoresent dyes
Real-Time PCR
uses probes or pieces of DNA or RNA that are labelled with a detectable molecule, such as biotin, dyes, or radioactive isotopes. These probes bind complementary to the target.
Probe Amplification Methods
examples of probe amplification methods
ligase chain reaction, cycling probe technology, cleavase invader
what are examples of amplification methods
target amplification
probe amplification
signal amplification
Relies on specific recognition and cleavage of particular DNA
structures by flap endonuclease-I family of DNA polymerases
Cleavase/Invader Technology
two primers are designed that hybridize target sequence in an
overlapping manner.
Cleavase/ Invader Technology
uses a stimuli to generate a signal, where the signal is proportional to the amount of the target sequence present in the clinical specimen assays
Signal Amplification Methods
examples are Branched DNA and Hybrid capture Assay
Signal amplification methods
a fully automated method, dsDNA is target of exponential amplification, amplifies target nucleic acid without use of thermocycler
Strand displacement amplification
an isothermal assay, targets either DNA or RNA ;
RNA is the amplified product, detection of M. tuberculosis
TMA or Transcription Mediate Amplification
similar to TMA but only RNA is targeted for amplification;
detection of HIV and CMV
NASBA or Nucleic Acid Sequence-Based Amplification
uses a collection of spots attached to a solid support that is capable of quantitating hundreds or even thousands of genes in a cell or a tissue simultaneously
Microarray or DNA Chip technology
separation of dsDNA to ssDNA and breaking of H bonds between base pairs
DENATURATION
between individual nucleotides or phosphate backbone; breaks in the backbone of DNA molecule either dsDNA or ssDNA
DEGRADATION
2 Causes of DNA Degradation
- Using very old DNA samples
- Using DNA extracted from formalin-fixed paraffin embedded samples
short term storage in weeks for DNA sample
4 C in Tris-EDTA
medium-term storage in months of DNA
-80 C in Tris-EDTA
Long-term storage in years under ethanol of DNA
-80 C as a precipitate
long term storage of DNA in decades
-164 C or dried
steps to prevent DNA degradation
- correct handling and storage
- perform extractions at 40C on ice or in the cold
- inhibit nuclease activity
- store purified DNA correctly
addition of phosphate groups is via
phosphoric anhydride linkage
examples of methods to determine nucleic acid purity and yield
- DNA Spectrophotometry
- Nanodrop Spectrophotometry
- Fluorometric Methods
- Real-Time PCR
Methods for Nucleic Acid Analyses
- Electrophoresis
- Hybridization Assay
- DNA Sequencing
- Polymorphism based
- Amplification Techniques
AGE means
Agarose Gel Electrophoresis
PAGE
Polyacrylamide Gel Electrophoresis
SDS-PAGE
Sodium dodecyl-sulfate Polyacrylamide gel Electrophoresis
DGGE
Denaturing gradient gel electrophoresis (DGGE)
TAE means
Tris-acetate-EDTA
TBE
Tris-Borate-EDTA
short nucleotide sequence complementary to a specific DNA sequence and initiate DNA replication
Primers
provides a free 3’-OH for DNA polymerase to start synthesis of chain
primers
use to target a particular sequence of complementary DNA or RNA ; can be labeled with radionuclide (P32), enzyme, and biotin
Probes
