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Flashcards in Exam 5: Molecular Medicine Deck (65):

adenosine deaminase (ADA)

absence of ADA enzyme causes severe immunodeficiency
required for breakdown of deoxyadenosine - without ADA deoxyadenosine accumulates and is converted to dATP (toxic)
Lymphocytes can't prevent dATP accumulation through dAMP degradation - leads to loss of lymphocytes and immune function
gene therapy to introduce wildtype ADA gene into lymphocyte cells



Prenatal diagnosis of genetic disease
Amniotic fluid is withdrawn with a needle at 15/16 weeks gestation.
Fluid contains fetal skin cells


Amplification Fragment Length Polymorphism (AFLP)

variation of length of amplified fragments detected during PCR
due to insertions or deletions between primer binding sites



molecule that induces an immune response
B-lymphocytes produce antibodies (bind to certain domain of antigenic agent) in response to antigen


artificial chromosomes

autonomously replicating gene delivery vectors for non-viral gene therapy
No insertion into genome required for stable expression, no size limit
However unpredictable chromosomal events happen during mitosis


autoimmune diseases

immune system falsely identifies proteins of body as antigens and attacks its own tissues and organs
fairly common (affect 5% of population)
Autoimmune antibodies can be detected by indirect ELISA


B-lymphocytes in antibody production

after detecting antigen, B-cells proliferate and produce antibodies that bind to antigen
Each B-cell produces only one type of antibody molecule


chorionic vilus sampling

for prenatal diagnosis of genetic disease
cells are taken from chorion with needle at 10-12 weeks gestation
cells are extraembryonic - chance of missing a mosaicism


Comparative Genome Hybridization (CGH)

used to detect copy number variation
uses DNA to detect insertions, deletions, and aneuploidies in the kilo to megabase range
single stranded DNA labelled with fluorescent dye & mixed and hybridized to metaphase chromosomes
CNV detected by uneven labeling of template chromosome (excess of patient fluorescence indicates duplication of pt's genome; excess in control genome indicate pt deletion)


complementary DNA (cDNA)

template for reverse-transcriptase PCR for RNA analysis
reverse transcription of RNA into cDNA by enzyme reverse transcriptase


copy number variation (CNV)

insertions or deletions cause structural variation in DNA, cell has a variation in number of copies of one or more sections of DNA
most are benign polymorphisms, but can cause disorders
Relatively small, so G-banding of metaphase chromosomes does not offer enough resolution to detect


cystic fibrosis (CF) gene therapy

in vivo gene therapy - deliver functional copy of CFTR gene into lung epithelial cells
adenovirus vector delivers gene into cells & induces production of CFTR protein
Needs frequent reapplication of therapeutic virus because cured cells recognized and destroyed by immune system



thousands of oligonucleotide spots can be printed on area of a square inch using low amounts of DNA (because amplification is required)
Can test for presence of all known mutant alleles of hundreds of different genes - allow genetic screening of pt for all known mutant alleles


epidermal growth factor (EGF) receptor

receptor protein-tyrosine kinase on cell surface coded by ErbB2 gene (overexpressed in some types of breast cancer) - tumor cells have a lot of EGF receptors
Antibody (Herceptin/Trastuzumab) binds to receptors and inhibits proliferation of tumors caused by HER2/Neu oncogene



part of antigen recognized by immune system, domain of antigen where antibodies bind


ex vivo gene therapy

manipulation of cells in gene therapy outside body
assumed safer than in vivo - effect of manipulation assessed in lab before inside pt
Cells have to be removed from pt, manipulated in lab, and re-implanted into pt
cells of epithelia or muscles cannot be harvested - can't use ex vivo gene therapy on these cells


Fluorescence in situ Hybridization (FISH)

allows identification of a chromosomal locus on a metaphase chromosome by hybridization with a specific fluorescent probe
Used to detect Copy Number Variations (CNV)


Genetic Information Nondiscrimination Act (GINA)

specifies that no individual can be discriminated against based on genetic information


genetic screening with microarrays

microarrays allow for screening of patient for all known mutant alleles in a single experiment


germ-line gene therapy

alter genome of reproductive cells
permanently alters genetic composition of individual and its offspring - prohibited because of ethical concerns



also known as ErbB2 gene - encodes for epidermal growth factor receptor
in 30% of breast cancers, ERB-2 gene is amplified and becomes an oncogene



antibody for therapy of metastatic breast cancer
binds to ErbB-2 receptors and inhibits proliferation of tumors caused by HER2/Neu
Also called Trastuzumab



Health Insurance Portability and Accountability Act protects privacy of medical records, including genetic data


HIV infection

if pt is infected with HIV, they will have antibodies against HIV-core proteins
these antibodies can be detected using indirect ELISA - viral core proteins bound to well serve as antigens which bind to HIV antibodies and change color in reaction well


hybridoma cells

created from fusion of B-lymphocyte fraction (post antibody stimulation) and myeloma cell line
immortal and produces antibodies
one hybridoma cell line selected for by quality and specificity of antibodies it produces and can be grown and used in clinical applications


immune response in gene therapy

body has a high antiviral defense system to prevent viruses from modifying genome
Use of viral gene delivery in gene therapy can be tricky - want to insert desired gene to as many cells as possible without being too aggressive and triggering a massive immune response


in situ hybridization

complementary DNA or RNA strand (probe) used to localize specific DNA or RNA sequence in a portion of tissue


in vivo gene therapy

aims to treat cells directly in patient
avoids harvesting and re-implanting cells
hard to reach all of target cells inside pt - works best on epithelial cells



lipid vesicles with DNA
fuse with cell membranes and release DNA into cells
Easy to use, commercially available, can accommodate large DNA molecules
However, have a slow entry into cells,& low integration rate of DNA into genome


nucleic acid hybridization

single strands of nucleic acids bind to other nucleic acid strands with complementary sequences
Probes with known sequences can be used to identify DNA or RNA molecules with complementary sequences



research to correlate genotypes and individual drug responses
differences in individual drug responses result from genetic polymorphisms
genes coding for drug targets determine how effective a drug inhibits the target enzyme; drug metabolizing enzymes determine how fast a drug is cleared from system


polymerase chain reaction (PCR)

amplification of DNA fragments
Uses DNA to detect small insertions and deletions
use oligonucleotide primers complementary to ends of DNA fragment to be studies - bind to single strand template DNA, carry out synthesis of complementary strand
Duplicates DNA fragment enclosed between primers & cycle is repeated about 30 times - billion fold amplification



small strands of DNA (oligonucleotides) used in PCR & ASO, anneal only to regions of very high sequence complementarity
DNA synthesized on template strand between primers


rheumatoid arthritis (RA)

autoimmune disease that arises from production of autoantibodies against immunoglobulin G
Can be diagnosed using indirect ELISAs


severe combined immunodeficiency

caused by absence of enzyme adenosine deaminase (ADA) - required for breakdown of deoxyadenosine
in lymphocytes ADA deficiency leads to accumulation of dATP (can't degrade dAMP) & loss of lymphocytes - leads to loss of immune function
first successful gene therapy trial treated inserted ADA gene into lymphocytes ex vivo


sickle-cell anemia mutations

most frequent cause is an A-T exchange in codon 6 of beta-globin gene
ASO is used to detect prevalent mutant alleles - DNA hybridized to wildtype and mutant-specific ASOs that are spotted on solid surface
Binding of labeled pt DNA to ASO-spots indicates presence of mutant or wildtype alleles - intensity of spots can be used to determine if pt is homozygous or heterozygous for mutant allele


SNP typing

Uses DNA to detect SNPs in entire genome
Only looks at regions of genome that are really different from one person to the next
looks at 10 million single nucleotide polymorphisms
pattern of SNPs gives information about individuals genetic characteristics


somatic gene therapy

repairing genome of differentiated cells in body


Gene therapy

insert functional copy of gene into mutant cells
requires mutation to be cured to be recessive & cells in question to be accessible to manipulation


stable expression of DNA in gene therapy

for effective integration of viral NA into host genome, genes delivered by virus need to be expressed stably
Requires integration of viral DNA into host genome, but not into random loci that could lead to cancer


Stability of Retrovirus

Stable, random insertion into genome


Adenovirus Stability

no integration, expression lost in 3-4 weeks


Adeno-associated virus stability



Herpes simplex virus stability

Stable, maintained outside of chromosome






most commonly used oral anticoagulant, causes severe bleeding in some pts if not dosed right
inhibits vitamin K epoxide reductase
response to warfarin determined by 2 enzymes of vitamin K metabolism:
polymorphism in detoxifying P450 protein (CYP2C9) - causes slow metabolism of warfarin (higher sensitivity)
mutation in VKORC1 subunit leads to higher tolerance


Western blotting

uses specific antibodies to indicate molecular weight of protein
electrophoresis of protein extracts - separates proteins according to molecular weight
transferred to membrane and incubated with specific antibody - bind to and identify protein of interest
position of protein on membrane indicates its molecular weight
Gives better information about antigen - size, isoforms than ELIZA which just sees quantification


Whole Exome Sequencing (WES)

uses DNA to detect point mutations/SNPs in exons - not introns or intergenic regions
codes for whole exon sequences (only 1% of genome, 30 million bp)
affordable technique, most relevant fraction of human genome


Whole genome sequencing

sequences complete human genome - 3 billion bp
problematic for sequencing of long stretches of repetitive DNA that have little or no function
neither feasible or useful in clinic


direct mutation detection

DNA sequencing
most useful in cases where exact nature of disease carrying mutation is known - may have novel, rare or unknown mutation that is not detected


indirect detection detection

used when exact mutation not known
follows an inheritance marker linked to mutant allele
problem - marker may have separated from disease by allele recombination


limits of DNA sequencing

Only show if person carries a mutation in heterozygous or homozygous form, but not which chromosome it is on
Can't deduce from sequencing data if SNPs are linked
Will not show if person has deletion or duplication of chromosomal region


PCR detects genetic polymorphisms by:

use of oligonucleotides that exclusively bind to either mutant or wildtype alleles - only those alleles will be amplified
Insertions or deletions detected using AFLP


Polyclonal antibodies

mixture of antibodies against different epitopes in antigen


monoclonal antibodies

cell line produce one specific antibody


enzyme-linked immunosorbent assay (ELISA)

detect and quantify antigens in vitro


sandwich ELISA

used to quantify antigen
antibody attached to well - in antigen is in sample, it will bind to antibody
second antibody against protein of interest is added and binds to antigen - attached to reporter molecule that fluoresces


sandwich ELISA used for

detection of peptides in human serum
Parathyroid Hormone, Glycated hemoglobin HBA1c, C-reactive protein (CRP)


indirect ELISA

used to detect presence of antibodies circulating in serum that shouldn't be there
antigen is attached to well - antibody binds to antigen & second reporter antibody is reactive against antibodies in general and fluoresces


indirect ELISA used for

HIV infection, autoimmune diseases, Rheumatoid arthritis


Direct sequencing

uses DNA to detect point mutations/SNPs


Reverse transcriptase PCR

uses RNA to detect expression levels for a small number of genes


Allele Specific Oligonucleotides (ASO)

uses DNA to detect point mutations, small insertions and deletions


Gene expression array

uses RNA to detect expression levels of thousands of genes - not inergenic regions or introns


Methylated DNA PCR

uses DNA to detect epigenetic changes, DNA methylation