Ch. 9. Molecular Diagnostics
I. Immunological Diagnostics II. Monoclonal Antibodies III. DNA Diagnostics and Forensic Analysis
I. Immunological Diagnostics
II. Monoclonal Antibodies
III. DNA Diagnostics and Forensic Analysis
Molecular Diagnostics
-Cancer -Infections caused by viruses, bacteria, fungi, parasites -Diseases caused by inheritance of mutant alleles Allele: different form of a gene or genetic locus e.g. sickle cell anemia, mutation in b-globin gene
-Cancer
-Infections caused by viruses, bacteria, fungi, parasites
-Diseases caused by inheritance of mutant alleles
Allele: different form of a gene or genetic locus e.g. sickle cell anemia, mutation in b-globin gene
Allele: different form of a gene or genetic locus
e.g. sickle cell anemia, mutation in b-globin gene
Two main approaches for developing diagnostic assays
1.) Immunoassay. Detection of antigens (cellular components: proteins, carbohydrates, metabolites) or antibodies associated with diseases (or physiological states, e.g. pregnancy) Exploits the specificity of binding of an antibody to an antigen 2.) DNA hybridization and PCR. Detection of DNA specific to a pathogen or a mutation that causes an inherited disease Exploits the specificity of binding of complementary DNAs or seecivicity of restriction enzymes
1.) Immunoassay.
Detection of antigens (cellular components: proteins, carbohydrates, metabolites) or antibodies associated with diseases (or physiological states, e.g. pregnancy) Exploits the specificity of binding of an antibody to an antigen
Detection of antigens (cellular components: proteins, carbohydrates, metabolites) or antibodies associated with diseases (or physiological states, e.g. pregnancy)
Exploits the specificity of binding of an antibody to an antigen
2.) DNA hybridization and PCR.
Detection of DNA specific to a pathogen or a mutation that causes an inherited disease Exploits the specificity of binding of complementary DNAs or seecivicity of restriction enzymes
Detection of DNA specific to a pathogen or a mutation that causes an inherited disease
Exploits the specificity of binding of complementary DNAs or seecivicity of restriction enzymes
Advantages
Sensitive: Uses small samples Rapid: Results within a few hours vs. several days for growth and identification of infectious agents Some pathogens can't be cultured or grow very slowly Simple: Often sold in kit form with premixed and standardized reagents Can be performed in clinics or out in the field (e.g. at home, villages, battlefield) rather than specialized laboatories Easy to automate Specific: Can be designed to detect a single infectious strain or genetic defect Safe: Culture of pathogens not required
Sensitive: Uses small samples Rapid: Results within a few hours vs. several days for growth and identification of infectious agents
Some pathogens can't be cultured or grow very slowly
Simple: Often sold in kit form with premixed and standardized reagents
Can be performed in clinics or out in the field (e.g. at home, villages, battlefield) rather than specialized laboatories Easy to automate
Can be performed in clinics or out in the field (e.g. at home, villages, battlefield) rather than specialized laboatories
Easy to automate
Specific: Can be designed to detect a single infectious strain or genetic defect
Safe: Culture of pathogens not required
Immune Response
Mammals and other higher organisms recognize foreign agents and substances as nonself Attempts to neutralize or destroy and eliminate them from the body
Mammals and other higher organisms recognize foreign agents and substances as nonself
Attempts to neutralize or destroy and eliminate them from the body
Immune system has two branches
1. Cell mediated immunity (carried out by cytotoxic T cells) 2. Humoral immunity (carried out by antibody secreting B cells) Most immunological diagnostic procedures use antibodies
1. Cell mediated immunity (carried out by cytotoxic T cells)
2. Humoral immunity (carried out by antibody secreting B cells)
Most immunological diagnostic procedures use antibodies
Terminology
Antibody: protein produce by a B cell; binds to antigens and targets them for destruction and elimination from the body
Antigen: foreign agent or substances that stimulate antibody production (Fig. 9.2)
Epitope: region of an antigen where a specific antibody binds (Fig. 9.2)
Some epitopes are more antigenic than others; stimulate a stronger immune response
Polyclonal antibodies: Produced by immune system in response to presence of an antigen
A mixture of antibodies that bind to the various epitopes of an antigen
Monoclonal antibody: antibody that recognizes a single epitope
See Fig. 9.1 For detection of an antigen in a sample (e.g. blood, urine, saliva, tissue) Color formation = target antigen present in sample Colorless = target antigen not present Similar idea to the immunoassay procedure we discussed in Chapter 4 for screening DNA libraries
See Fig. 9.1
For detection of an antigen in a sample (e.g. blood, urine, saliva, tissue)
Color formation = target antigen present in sample
Colorless = target antigen not present
Similar idea to the immunoassay procedure we discussed in Chapter 4 for screening DNA libraries
Polyclonal vs. Monoclonal Antibodies for Testing
Polyclonal or monoclonal antibodies may be used as diagnostics, but:
Monoclonals usually preferred when human health is involved. Fewer false positive or false negative test results
Monoclonals usually preferred when human health is involved.
Fewer false positive or false negative test results
Production of Monoclonal Antibodies
Fig. 9.4 Produced by cultured cells rather than by an animal B cells cannot be grown in cell culture for antibody production, however Hybridomas: fusion of B cells with immortal myeloma cells (cancerous B cells) can be cultured for production of monoclonal antibodies Hybridomas producing a single antibody specific for an epitope (monoclonal antibody) must be isolated HAT medium is used to select for growth of hybridomas Hybridomas are isolated and screened to identify the one that produces a useful monoclonal antibody
Fig. 9.4
Produced by cultured cells rather than by an animal
B cells cannot be grown in cell culture for antibody production, however
Hybridomas: fusion of B cells with immortal myeloma cells (cancerous B cells) can be cultured for production of monoclonal antibodies
Hybridomas producing a single antibody specific for an epitope (monoclonal antibody) must be isolated
HAT medium is used to select for growth of hybridomas Hybridomas are isolated and screened to identify the one that produces a useful monoclonal antibody
HAT medium is used to select for growth of hybridomas
Hybridomas are isolated and screened to identify the one that produces a useful monoclonal antibody
Diagnostic monoclonal antibodies have been produced for detection of a variety of antigens (Fig. 9.5)
DNA-Based Diagnostics
Exploits specificity of:
1. Binding of labeled DNA probes to a complementary target DNA sequence (Fig. 9.6)
Ex. Gene unique a pathogen Gene with a genetic defect (mutation) Methods for detection of label -Radioactivity -Fluorescence -Colored product -Light (Chemiluminescence)
Ex.
Gene unique a pathogen Gene with a genetic defect (mutation)
Gene unique a pathogen
Gene with a genetic defect (mutation)
Methods for detection of label
-Radioactivity
-Fluorescence
-Colored product
-Light (Chemiluminescence)
2. Binding of PCR primers to target sequence
3. Sequence of DNA recognized by restriction enzymes
Detection of Labeled Probes
I. Direct detection of probe label
1. Radioactivity 32P or 35S labeled probes Detect by autoradiography 2. Fluorescence Probe is labeled with a molecule that fluoresces Detect by illuminating with light that causes molecule to fluoresce
1. Radioactivity
32P or 35S labeled probes Detect by autoradiography
32P or 35S labeled probes
Detect by autoradiography
2. Fluorescence
Probe is labeled with a molecule that fluoresces Detect by illuminating with light that causes molecule to fluoresce
II. Indirect detection. DNA label is bound by an enzyme with a detectable activity
Enzyme may be attached to an antibody or to biotin 1. Chemiluminescence Enzyme converts a substrate to a product that emits light Detect by autoradiography
Enzyme may be attached to an antibody or to biotin
1. Chemiluminescence
Enzyme converts a substrate to a product that emits light Detect by autoradiography
Enzyme converts a substrate to a product that emits light
2. Color Enzyme converts a substrate to a colored product Detect visually or by absorption of light (spectrophotometry)
2. Color
Enzyme converts a substrate to a colored product Detect visually or by absorption of light (spectrophotometry)
Enzyme converts a substrate to a colored product
Detect visually or by absorption of light (spectrophotometry)
Use and Indirect Detection of a Biotinylated DNA Probe
Fig. 9.7 Probe is labeled with biotin Enzyme also attached to biotin
Fig. 9.7
Probe is labeled with biotin
Enzyme also attached to biotin
Use of DNA Probes for Detection of Genetic Defects
PCR/OLA -Polymerase Chain Reaction/Oligonucleotide Ligation Assay (Fig. 9.18)
For detection of point mutations PCR amplifies the region where the mutation occurs to increase sensitivity OLA determines if the mutation is present
For detection of point mutations
PCR amplifies the region where the mutation occurs to increase sensitivity
OLA determines if the mutation is present
Ex. Detection of carriers of sickle cell anemia (heterozygotes) (Fig. 9.17) Mutant allele eliminates one restriction site for Cvn I in the b-globin gene
Ex. Detection of carriers of sickle cell anemia (heterozygotes) (Fig. 9.17)
Mutant allele eliminates one restriction site for Cvn I in the b-globin gene
Detection of more than one mutantion in a gene
PCR amplifies DNA in all regions where known mutations occur Primers label the products with biotin DNA probes that are perfectly complementary to the mutant sequences are bound to a membrane PCR products are hybridized to the probe DNA on the membrane If mutation is present, hybridization occurs and is detected colorometrically using alkaline phosphtase conjugated to streptavidin
PCR amplifies DNA in all regions where known mutations occur
Primers label the products with biotin
DNA probes that are perfectly complementary to the mutant sequences are bound to a membrane
PCR products are hybridized to the probe DNA on the membrane
If mutation is present, hybridization occurs and is detected colorometrically using alkaline phosphtase conjugated to streptavidin
Other Applications of DNA Detection
See Fig. 9.12 DNA fingerprinting for forensic analysis or determining paternity of a child Determines if two samples containing DNA are from same source Ex.. Crime suspect's DNA and DNA from crime scene 1. Digest DNA samples with a restriction enzyme 2. Analyze by electrophoresis to separate DNA fragments by size 3. Transfer fragments to a membrane and hybridize to labeled DNA probes (Southern blot) 4. Compare the patterns (DNA fingerprints) produced by the suspect and evidence Based on variation in length of DNA fragments due to presence of repetitive sequences Ex. GATA may be repeated 5 times in the victim, 8 times in the suspect and 5 times in blood on the suspect's shirt. The suspect probably made the victim bleed.
See Fig. 9.12
DNA fingerprinting for forensic analysis or determining paternity of a child
Determines if two samples containing DNA are from same source
Ex.. Crime suspect's DNA and DNA from crime scene
1. Digest DNA samples with a restriction enzyme
2. Analyze by electrophoresis to separate DNA fragments by size
3. Transfer fragments to a membrane and hybridize to labeled DNA probes (Southern blot)
4. Compare the patterns (DNA fingerprints) produced by the suspect and evidence
Based on variation in length of DNA fragments due to presence of repetitive sequences
Ex. GATA may be repeated 5 times in the victim, 8 times in the suspect and 5 times in blood on the suspect's shirt. The suspect probably made the victim bleed.
Ex. GATA may be repeated 5 times in the victim, 8 times in the suspect and 5 times in blood on the suspect's shirt.
The suspect probably made the victim bleed.
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