1050 Unit 3 Flashcards
Describe affinity
-initial attraction force between a single Fab site on an antibody molecule and a single epitope of an antigen
—->strength of attraction depends on specificity of antibody for antigen
—->cross reacting antigens have lower affinity
Describe avidity
-sum of attractive forces between an antigen and antibody
—->strength with which a multivalent antibody binds a multivalent antigen
—->measure of the overall stability of an antigen antibody complex
describe how the law of mass action relates to antigen-antibody binding
-value of K depends on strength of binding between antibody and antigen
-the higher the value of K–>larger the amount of antigen-antibody complex –> the more visible or easily detectable the reaction.
define precipitation
the combination of soluble antigen with soluble antibody to produce insoluble complexes that are visible
define agglutination
process by which particulate antigens (latex beads, RBC, gel particles) react with specific antibody to form large aggregates or clumps
what is the difference between precipitation and agglutination?
agglutination takes place between antibody and particular antigen where as precipitation happens between antibody and soluble antigens
describe antigen/antibody concentration in prozone
-when antibody excess is large, prozone occurs
-antigen combines with only one or two antibody molecule and no cross linkage formed.
-precipitation and agglutination can not be detected
describe antigen/antibody concentration in postzone
-when antigen excess is large, postzone occurs
-small aggregates are surrounded by excess antigen. Every available antibody site is bound to single antigen
describe antigen/antibody concentration in zone of equivalence
-number of multivalent sites of antigen and antibody molecules is approximately equal
describe immunoturbidity
-measures reduction in light intensity (measurement of turbidity)
-spectrophotometer
describe nephelometry
-measures light scatter at particular angles as immune complexes form
-nephelometer
-amount of light is proportional to size, shape, and concentration of molecules. Thus light scatter increases as number of immune complexes increases and is an index of concentration index of antibody or antigen is solution.
what is single diffusion?
involves migration of antigen only.
what is double diffusion?
both antigen and antibody diffuse independently through a semisolid medium in two dimensions, horizontally and vertically
what is the principle of the end point method of radial immunodiffusion (RID)?
-antigen is allowed to diffuse to completion when equivalence is reached
-single diffusion technique
-incorporated into gel
- antigen is placed on gel in wells which diffuse out and react with antibody, forming rings of precipitation around the wells. Diameter of ring is directly related to the amount of antigen in the well.
describe the principle of immunofixation electrophoresis (IFE)
-proteins in patients serum are electrophoresed, then antibody is applied directly to gel (agarose or cellulose acetate gel). Precipitation forms where antigen-antibody combination has taken place
-used to detect monoclonal immunoglobulins produced by patients with immunoproliferative diseases such as multiple myeloma
discuss IgM’s ability to participate in agglutination reactions
-700 times more efficient at agglutination than IgG
-bigger, which contributes to its ability to agglutinate without enhancement
-tested at room temperature
discuss IgG’s ability to participate in agglutination reactions
- usually requires use of enhancement techniques to achieve visible reaction such as ionic strength of solution, pH and temperature
-usually requires the use of second antibody to visualize reaction–> Coombs reagent
-tested at 37C
what are the physiological conditions that can be altered to enhance agglutination
-pH
-Temperature
-ionic strength of solution
describe Agglutination inhibition
- based on competition between antigen coated particles and soluble patient antigen for a limited number of antibody site
-only instance in which agglutination represents a negative test.
define Agglutinins
Antibody, lectin or other substances that causes agglutination
define Coombs reagent
Poly alert and contains species specific anti-IgG, anti-IgM, and anti- c antibodies. If present on patient RBC, cross linking will occur (agglutination)
Define cross reactivity
Reacting of an observed agent which initiates reactions outside the main reaction expected
Define Electrophoresis
Movement of charged particles in a fluid or gel under the influence of an electric field
Define end point method
Measure total amounts of analytes that participate in the reaction
Describe DNA
-made of nucleotides that contain deoxyribose sugar with one of the following: adenine, thymine, cytosine, and guanine
-double stranded and arrange in a double helix
What happens when DNA seperates?
two daughter strands separate; each is a template for a newly synthesized complementary strand
Describe RNA
-nucleotides that contain ribose sugar and the nitrogen bases: adenine, uracil, guanine and cytosine
- single stranded
what is the basis of all molecular diagnostic testing?
the high specificity of detection of nucleic acid sequences through complementary base pairing
what does “central dogma of molecular biology refer to?
-the fact that DNA serves as the template for messenger RNA, which in turn codes for proteins
explain mutations and polymorphisms
-changes in nucleotide sequences that may affect specific protein structure and function
explain technique of gel and capillary electrophoresis
negatively charged DNA fragments are separated by size under the force of an electric current in a semisolid gel or polymer solution
Define Restriction fragment length polymorphisms
-(RFLP)
-change in DNA that result in different size pieces when cleaved by restriction enzymes
What is the CRISPR-Cas9?
gene editing system that can be used to alter DNA at specific location
define hybridization
very specific binding of two complementary DNA strands or a DNA and a RNA strand. Often a probe is used to detect an unknown nucleic acid sequence in sample
what are some hybridization techniques?
1) Southern blot analysis
2) situ hybridization
what is amplification?
involves making many copies of specific nucleic acid sequence to obtain enough material for laboratory identification
describe transcription-mediated amplification
-TMA
-target is RNA instead of DNA. cDNA copy is made of the original RNA and uses to produce millions of RNA copies
describe strand displacement amplification
-SDA
-involves amplification of probes rather than the original DNA
describe Branched DNA
represents a signal amplification method in which multiple probes attach to the original target sequence
-DNA probes used to capture the target nucleic acid
what are types of probe amplifications?
1) strand displacement amplification (SDA)
2) loop-mediated amplification (LAMP)
3) molecular inversion probe (MIP) method
what does DNA sequencing involve?
-determining the order of nucleotides in a DNA chain -the most specific way of detecting polymorphisms and mutations
What is the Sanger chain termination sequencing method?
-involves replicating a single DNA strand in the presence of fluorescent-labeled modified nucleotide bases called dideoxy nucleotide triphosphates
-DNA fragments of various sizes are generated from the original template, and the sequence is determined by detecting the fluorescent labels
-most explicit method for identifying polymorphisms and mutations
what is pyrosequencing?
alternate method that relies on the generation of the light when nucleotides are added to a growing DNA chain
-no gels, dyes or ddNTPs
what is the overall function of Next Generation Sequencing?
-NGS
-allows for rapid sequencing of large number of small DNA templates at one time. The short sequences are then assembled into a complete sequence.
what does targeted gene panels do?
determine the sequence of specific genes
what does whole exome sequencing do?
sequence of only the coding regions within DNA
what does whole genome sequencing do?
sequence of an entire genome to identify mutations associated with a variety of diseases
what is bioinformatics?
uses information technology to analyze the vast amount of data for clinical relevance by comparison with known databases
what is associated only with RNA synthesis?
promoter
the speed at which nucleic acids migrate in gel electrophoresis is determined by which property?
size
what is the function of endonucleases?
they cleave DNA at specific site
what technique is used in RNA-guided enzymes?
CRISPR
To what does situ hybridization refer to?
probes react with the intact cells within tissues
what is the principle of microarrays?
arrays contain multiple unlabeled probes on a solid support
Describe PCR
primers are used to make multiple DNA copies
what happens in the annealing process of PCR?
the primers bind to target DNA
what is the purpose of the amplification control in qPCR?
to avoid false negatives
what technique is based on RNA amplification?
TMA (target mediated)
what is used in Sanger sequencing?
ddNTP
what type of signal is generated in pyrosequencing?
light
what is the NGS sequencing library?
a collection of short templates to be sequences simultaneously
what is the coverage in NGS?
the number of times a region is sequenced
define nucleic acid
carry genetic information that codes protein structure
what binds Guanine to Cytosine
3 hydrogen bonds
what binds adenine and thymine?
2 hydrogen bonds
what are DNA and RNA measured in?
-DNA is measured in base pairs
-RNA is measured in bases
what is a chromosome?
double helix of DNA
how many chromosomes are in the cell nucleus?
46
Define genes
sequences of nucleotides in chromosomes that carry information for either a protein or non-coding RNA molecule
Define diploid
two copies of the each 23 chromosomes per cell or 46 chromosomes total
define genome
entirety of of DNA in cell
what is the cell cycle and what are their phases?
as cells divides, they undergo a series of events in which they increase in size and duplicate their DNA
-phases
—1> G1
—2> S
—3> G2
—4> mitosis/cytokinesis
explain each phase from the cell cycle
- G1–> daughter cell will go/be here
- S –> DNA replication takes place
- G2–> DNA complement of the cell is doubled
-mitosis - one complement of chromosome is divided into two daughter cells
what is the catalyst for DNA replication?
DNA polymerase enzyme
what can DNA synthesis not start without
a preexisting 3’ hydroxyl group
what is a primer?
-begins DNA synthesis in vivo
- a primer of RNA is synthesized by RNA polymerase (primase) enzyme
what does PCR stand for?
polymerase chain reaction
what is the lagging stand of DNA?
copied discontinuously toward replication fork
what is the leading stand of DNA?
copied continuously is direction of replication
what is RNA synthesis catalyzed by?
RNA polymerase
What is RNA polymerase?
begins polymerization of RNA by binding to its binding recognition start site in DNA (promoter)
Define epigenetics
-involves chemical changes is histone proteins
-modification of DNA such as base methylation
-noncoding RNA activities that can influence the expression of genes independent of the nucleotide sequences