MODX Flashcards
Attachment of the 5’ phosphate and hydroxyl groups of an incoming nucleotide to the 3’ hydroxyl group of the last nucleotide on the growing chain
Nucleic acid chain
A macromolecule made of nucleotides bound together by the phosphate and hydroxyl groups on their sugars
Nucleic acid
DNA is oriented in a
5’ to 3’ directions
A macromolecule of carbon, nitrogen, oxygen, phosphorous, and hydrogen atoms
DNA
4 nitrogen bases that makes up the majority of DNA
Adenine, cytosine, guanine, thymine
Nitrogen bases are attached to a ____ sugar, which forms a polymer with the deoxyribose sugars of other nucleotides through a ______
Deoxyribose sugar, phosphodiester bond
Published a paper on NUCLEIN in 1871
Johann Friedrich Miescher
First one who describe the double helical stucture of DNA
James watson and Francis Crick
Each nucleotide consists of a five-carbon sugar, the first carbon of which is covalently joined to a ______ and the fifth carbon to a _____
Nitrogen base, phosphate moiety
The nitrogen base bound to an unphosphorylated sugar is
Nucleoside
Nucleoside is composed of
Adenosine, guanosine, cytidine, and thymidine
Nucleotides can be converted to nucleosides by
Hydrolysis
Important for forming the phosphodiester bond that is the backbone of the DNA strand
Hydroxyl group on the third carbon
Are planar carbon-nitrogen ring structures
Nitrogen bases
Four common nitrogen bases in DNA
Adenine, Guanine, Cytosine, and Thymine
Nitrogen bases with a SINGLE ring structure are called
Pyrimidines (Thymine, cytosine)
Bases with DOUBLE ring structures are called
Purines (Guanine, adenine)
The key to the specificity of all nucleic acid-based tests used in the molecular laboratory
Hydrogen bonds
How the information held in the linear order of the nucleotides is maintained
Specific Hydrogen bond formation
The formation of hydrogen bonds between two complementary strands of DNA is called
Hybridization
The double helix are identical not complementary
True or false
FALSE
The double helix are complementary not identical
DNA has an ____ orientation because of the way it is replicated
Antiparallel orientation
Products of transcription and translation of the nucleic acid
Proteins
Defined as the ordered sequence of nucleotides on a chromosome that encodes a specific functional product
Gene
Translation takes place on
Ribosomes
DNA transcribed and then translated into protein, this process is called
Gene expression
Copying of one strand of DNA into RNA by a process similar to that of DNA replication
Transcription
Transcription is catalyzed by
RNA polymerase
Transcription occurs in
Interphase
Prokaryotes ribosome is 70s
70S Ribosomes is composed of
30S small subunit and 50S large subunit
The 30S subunit is composed of? And what is it’s weight
1 million daltons
composed of: 16S ribosomal RNA and 21 ribosomal proteins
What is the weight of the 50S subunit and what is its composition
1.8 Million daltons
Composed of 23S Ribosomal RNA and 34 ribosomal proteins
Eukaryotic ribosome = 80S
80S is composed of
40S small subunit and 60S Subunit
The 40S small subunit from eukaryotes is composed of? What is its weight
1.3 million daltons
Composed of 18S Ribosomal RNA and 30 ribosomal proteins
The 60S subunit from eukaryotes is composed of? What is its weight
2.7 million daltons
Composition:
5S Ribosomal RNA, 5.8S Ribosomal RNA, 28S ribosomal RNA and about 40 ribosomal proteins
Protein synthesis in the ribosome almost always starts with
The amino acid methionine in eukaryotes and N-formylmethionine in bacteria and chloroplasts
First peptide bond is formed between the amino acids in the A and P sites by transfer of the _____ group of the first amino acid to the amino group of the second amino acid, generating a dipeptidyl-tRNA in the A site
N-formylmethionyl
This step is catalyzed by an enzymatic activity in the large subunit called
Peptidyl transferase
During translation, the growing polypeptide begins to fold into its mature conformation. This process is assisted by molecular ____
chaperones
These specialized proteins bind to the large ribosomal subunit, forming a hydrophobic pocket that holds the emerging polypeptide
Molecular chaperones
Protects the growing unfinished polypeptides until they can be safely folded into mature protein.
Chaperones
E. coli can synthesize a ___ - ___ amino acid protein in 10-20 seconds
300-400 amino acid protein
In nucleated cells, the majority of translation occurs in
the Cytoplasm
Purpose of nucleic acid extraction
To release the nucleic acid from the cell for use in subsequence procedures
Should be free of contamination with protein, carbs, lipids, or other nucleic acid
The initial release of the cellular material is achieved by breaking the cell and nuclear membranes also known as
Cell lysis
Following lysis, the target material is ___, and then the concentration and purity of the sample can be determined
Purify
A highly branched sucrose polymer that does not penetrate biological membranes
Ficoll
Least damaging among tested fixatives
Buffered formalin
Less toxic xylene substitutes
Histosolve, Anatech Pro-Par, or ParaClear
More rapid and comparably effective DNA extraction can be performed using solid matrices to bind and wash the DNA
Solid-Phase Isolation
The carrier in solid-phase separation requires the nucleic acid of more than ____ nucleotides in length
200
Promega DNA IQ holds a specific amount of DNA. What is the amount?
100ng
Will digest proteins in the sample, lysing the cells and inactivating other enzymes
Proteinase K
A cation-chelating resin that can be used for simple extraction of DNA
Chelex (Chelating resin)
Most commonly used in forensic applications but may also be useful for purification of DNA from clinical samples and fixed, paraffin-embedded specimens
Extraction with Chelating Resin
Provides sufficiently clean DNA that can be used for amplification
DNA extraction/storage card
RNA is labile due to the ubiquitous presence of
RNAses
Small proteins that can renature, even after autoclaving, and regaining activity
RNAses
T/F
RNAses must be eliminated or inactivated before isolation of RNA
True
Glassware must be baked for 4 to 6 hours at ____C to inactivate the RNAses
400C
Most abundant RNA in all cells
Ribosomal RNA
2nd most abundant RNA
Messenger RNA
Reticulocytes in blood and bone marrow samples are lysed by ____ or separated from WBCs by ___
Lysed by osmosis, separated from wbcs by centrifugation
Organic method of extraction
Phenol - Chloroform
Inorganic method of extraction
Salt precipitation to remove proteins
Solid phase method of extraction
Column Filter, magnetic beads
Purification of DNA
DNA precipitated by alcohol
RNAses
Ubiquitous
Very high concentration on hands
Act at wide range of temperatures: -20 to >100C
Special considerations for RNA isolation
Storage: Liquid nitrogen or at -80C
Gloves should always be worn because hands have high concentration of RNAse
Use equipment dedicated to RNA testing
Reserve areas in the laboratory for storage and RNA work
Use disposable items direct from manufacturer
Avoid reusable glassware or bake 4-6 hours at >270C to inactivate RNAse
Uses RNAse inhibitors during cell lysis e.g guanidine thiocyanate
Hydrophobic portion of organicmethod
Lipids and debris on the bottom
Hydrophilic portion of organic method
aqueous phase contains the DNA/RNA on top of isopropyl alcohol
low pH and high salt precipitate proteins leaving DNA in solution (Supernatant)
Inorganic method
1 million eukaryotic cells or 10 to 50mg of tissue will yield how many amounts of RNA?
10ug of RNA
Fluorescent dyes that binds specifically to DNA are used to visualize the sample preparation
Ethidium bromide or SybrGreen I
SybrGreen II are used to detect
Single stranded DNA or RNA
Used to detect small amounts of DNA by visual insepction
Silver stain
Nucleic acids absorb light at
260nm
Phenol absorbs ultraviolet light at
270nm to 275nm
May be used to estimate the purity of nucleic acid
Spectrophotometric measurement
Common contaminants and their wavelengths of peak absorbance
Organic compounds = 230nm
Phenol = 270nm
Protein = 280nm
Particular matter = >330nm
Absorbs light at 280nm through the aromatic tryptophan and tyrosine residues
Proteins
The absorbance of nucleic acid at 260 nm should be
1.6 to 2.00 times more than the absorbance at 280nm
Early methods use this. A dye that combines with alpha methylene aldehydes/Ribosomes to yield a fluorescent product
3,5-Diaminobenzoic acid 2HCl DABA
This dye combines with adenine-thymine base pairs in the minor groove of the DNA double helix and is thus speicific for intact double-stranded DNA
Hoechst 33258
Other DNA-specific dyes that can be used for fluorometric quantification
PicoGreen and OliGreen
More sensitive than Hoechst dye due to brighter fluorescence upon binding to double-stranded DNA
PicoGreen
Designed to bind to short pieces of Single-stranded DNA
OliGreen
A microfluidics-based instrument used for lab on a chip technology that has been applied to nucleic acid quantification and analysis
Agilent 2100 Bio-analyzer
Useful for analysis of studies on small RNAs, such as mucroRNAs in eukaryotes and gene expression in bacteria
Microfluidics
Used in Horizontal orientation electrophoresis
Polysaccharide extracted from seaweed
Easy and cost-effective
Larger fragments (20bp - 10Mb)
Agarose Gel
Used in vertical orientation electrophoresis
Synthetic material
Higher resolution, finer matrix, higher resolution capability
Dangerous neurotoxin
Smaller sequences/fragments
Polyacrylamide gel
Choice of agarose concentration for DNA cells
0.3 = 5000 - 60,000 bp
0.6 = 1000 - 20,000 bp
0.8 = 800 - 10,000 bp
1.0 = 400 - 8,000 bp
1.2 = 300 - 7,000 bp
1.5 = 200 - 4,000 bp
2.0 = 100 - 3,000 bp
Choice of polyacrylamide concentration for DNA cells
3.5 = 100 - 1,000 bp
5.0 = 80 - 500 bp
8.0 = 60 - 400 bp
12.0 = 40 - 200 bp
20.0 = 10 - 100 bp
Monitors the progress of the electrophoresis run as the dye approaches the end of the gel
Runs ahead of the smallest fragment of DNA; not associated with the sample DNA
Example is Bromophenol blue, Xylene cyanol
Tracking Dye
Increases the density of the sample as compared with the electrophoresis buffer
Density gradient examples are: Ficoll, Sucrose, Glycerol
Loading Dye
Compare the nucleic acid fragments with a ladder
Molecular weight size marker
Set of standards that are used to identify the approximate size of a molecule on a gel during electrophoresis
Mixture of fragments with known size to compare the PCR products (Amplicons)
Ladder
Agarose gel is comprised of
Agaropectin and agarose
Used for very large pieces of DNA (50,000 - 250,000 bp)
Pulse field Gel electrophoresis
Works by alternating the positive and negative electrodes during electrophoresis
Requires temperature control and a switching mechanism
Field Inversion Gel Electrophoresis
Very small DNA fragments and single-stranded DNA are best resolved on?
Polyacrylamide gels in polyacrylamide gel electrophoresis
Acrylamide in combination with the cross-linker methylene bisacrylamide
Originally applied mostly for protein separation
Used for sequencing nucleic acids, mutation analyses, nuclease protect assays
Polyacrylamide gels
Catalyst of polyacrylamide gel to polymerize it
Ammonium persulfate + Tetrametyhlethylenediamine
or
Light activation
Widest application of this is the separation of such organic chemicals such as pharmaceuticals and carbohydrates
Applied to the separation of inorganic anions and metal ions
Faster analytical runs and lower cost per run than HPLC
Used for the separation and analysis of nucleic acid
Capillary electrophoresis
Analyte is resolved in a thin glass capillary (Fused silica)
Fused silica is covered with a polyimide coating for protection
Capillary electrophoresis
Used for nucleic acid analysis
The platinum electrode close to the end of the capillary undergoes a transient high-positive charge to draw the sample into the end of the capillary
Electrokinetic injection
Other application of capillary electrophoresis
Clonality testing, microsatellite instability detection, bone marrow engraftment analysis
Advantage of capillary system over traditional slab gel electrophoresis
Increased sensitivity and immediate detection
Purpose of this is to carry the current and protect the samples during electrophoresis
Buffer system
Most common buffer for electrophoresis
Tris borate EDTA (TBE)
Tris Phosphate EDTA (TPE)
Tris acetate EDTA (TAE)
T/F
TBE has a greater buffering capacity than TAE
True
DNA will migrate twice as fast in ___ than in TBE in constant current
TAE
Not recommended for some post electrophoretic isolation procedures
TBE
Modifies sample molecules in ways that affect their migration
Buffer additives
Example of buffer additinves
Formamide, urea, and various detergents
Breaks hydrogen bonds between complementary strands or within the same strand of DNA or RNA
Denaturing agent
___ and heat added to DNA and RNA breaks and blocks the hydrogen bonding sites, hindering complementary sequences from reannealing
Formamide and heat
Denaturant used for RNA that reacts with amino groups on the RNA to prevent base pairing between homologous nucleotides and with aldehydes, which also disrupt base pairing
Methylmercuric Hydroxide (MMH)
Examples of RNA buffers
Sodium phosphate,
MOPS buffer propanesulfonic acid
Sodium acetate
Decrease of pH at the Cathode and increase of the pH at the anode is called
pH Drift
What affects the current in electrophoresis
Thickness of the gel and the volume of the buffer
Horizontal gels are also called
submarine gels
Inserted into the top of the gel to create holes or wells
Comb
Slowing migration in the outer lanes compared with lanes in the center of the gel due to the outer gel being cooler than the middle gel is called
Gel smiling
What is the size of the spacers of the vertical gel
0.05mm to 4mm
that are placed upside down, teeth up, not in contact with the gel to form a trough on the gel during polymerization is called
Wells can be loaded while this comb is in place
Advantage of this comb is the lanes are placed immediately adjacent to one another
Shark tooth’s comb
SyBr Gold is used for
Both DNA and RNA staining
Difference between Sybr green 1 and EtBr
Sybr green 1 differs from EtBr in that it does not intercalate between bases
In agarose gel, SyBr staining is ____ times more sensitive than EtBr
25-100
Originally developed for protein visualization
Sample is fixed with methanol and acetic acid
Silver stain
Silver stain:
Best for thick gels -
More stable -
Silver stain:
Best for thick gels - Silver diamine
More stable - Silver nitrate
Involves making copies of a target sequence to such a level that they can be detected in vitro
Target amplification
Visualized the idea of amplifying DNA in vitro in 1983
Kary Mullis
Real advantage of PCR
Ability to amplify specific targets
Accomplished by heating the sample at 94C to 96C for several seconds to minutes.
Turns double stranded DNA into two single strands
Denaturation
2nd and most critical step for the specificity of the PCR
Temperature range at 50C to 70C
Annealing
Third and last step of the PCR cycle
The polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers
Optimal temp for this step is 68C to 72C
Primer Extension step
The critical component of the PCR because they determine the specificity of the PCR
Primers
The building blocks of DNA
Nucleotide triphosphates
Automation of the PCR procedure was greatly facilitated by the discovery of the thermostable enzyme called
TAQ polymerase
From thermus thermophilus
Tth Polymerase
Fragment that lacks the N-terminal 289 amino acids of Taq polymerase and its inherent 3’ to 5’ exonuclease activity
Stoffel Fragment
Provides the optimal conditions for enzyme activity
PCR buffer
Binds inhibitors and stabilizes the enzyme
Bovine serum albumin
Provides reducing conditions that may enhance enzyme activity
Dithiothreitol
Lower the denaturing temperature of DNA with high secondary structure, thereby increasing the availability for primer binding
Formamide
Designed to rapidly and automatically ramp to the required incubation temperature. holding at each one for designated periods
Thermal cyclers or thermocyclers
Work with small sample volumes in chambers that can be heated and cooled quickly by changing the air temperature surrounding the samples
Rapid PCR
Equipped with fluorescent detectors to measure PCR product as the reaction proceeds
Real-time PCR
Isolated from the bacterium Thermus aquaticus
TAQ DNA polymerase
Complementary DNA is first produced from RNA targets by reverse transcription, and then the cDNA is amplified by PCR
Requires primers such as oligo dT primers or random hexamers to prime the synthesis of the initial DNA strand
Reverse-transcriptase PCR
Developed to increase both the sensitivity and specificity of PCR
Uses two pairs of amplification primers and two rounds of PCR
One primer pair is used in the first round of the amplification of PCR of 15-30 cycles. The products of the first round of amplification are then subjected to a second round of amplification using the second set of primers
The second set of primers anneal to a sequence internal to the sequence amplified by the first primer set
Nested polymerase Chain Reaction
Two or more primer sets designed for amplification of different targets are included in the same PCR reaction
Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube
Multiplex polymerase chain reaction
Can be accomplished by capturing or isolating each individual nucleic acid molecule present in a sample within many chambers, zones, or regions that are able to localize and concentrate the amplification product to detectable levels
Detection and quantification of low levels of pathogen sequences, expression of rare genetic sequences in single cells, and clonal amplification of nucleic acid for sequencing mixed nucleic acid samples
Digital polymerase chain reaction
Detects the accumulation of amplicon after each thermal cycle in real time
qPCR/Real-time pcr
Inhibits enzymes used in molecular analysis such as reverse transcriptases and DNA polymerases in vitro
Heparin and occasionally ACD
Fresh specimen
Fluorescent in situ hybridization (FISH) and karyotyping
A specially designed labels are available for long-term nucleic acid storage at ultra-low temparature
Cryotubes
Refrigeration of blood will cause neutrophils to
degranulate, releasing enzymes that affect free virus particles
Storage for viral RNA in whole blood
22-25 C (Room temp)
Isolated DNA of sufficient purity can be stored at room temp for
Several months at rt or at least 1 year in the refrigerator
Purified DNA can be stored at
Freezer temp -20C to -70C in tightly sealed tubes for up to 10 years or longer
Serves as internal controls in FISH analyses
Centromere-specific probes
Monitored daily
Refrigerator and freezers
Thermometers used in monitoring thermal cyclers
With flexible probes
Type K thermocouples
Monitored at least annually using tachometer
Centrifuges and microcentrifuges
Tested annually to ensure delivery of accurate voltage and current
Power supplies
Background measurements are required on such instruments as
Fluorometric detectors, luminometeres, and denitometers
Maintenance includes scanning through the range of wavelengths used (200 to 800nm)
Must be check annually
Spectrophotometers
Monitored annually for proper air flow
Fume hoods and laminar flow hoods
Checked for accuracy before use and at 6-month interval
Pipettors
Most conveniently supplied in lyophilized (freeze-dried) from the DNA synthesis facilities
Primers
Probes, primers, antibodies, and other test components that detect a specific target
Analyte-specific reagents
MOST asr’s used in the molec lab are class _
Class I
Class II and III ASRs include those used by
Blood banks to screen for infectious diseases and those used in diagnosis of certain contagious diseases
Use in the diagnosis of a specific disease or other condition
In vitro diagnostic
Refers to the analysis of external specimens supplied to independent laboratories from a reference source
Performed atleast twice a year
Proficiency testing
