Introduction to molecular diagnostics Flashcards by Iseoluwa Ojo

____________ are the foundation of a successful health care system, providing critical information that health care providers and patients need to make the right medical decisions.

Diagnostic tests

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Diagnostics often provide objective, quantitative measurements that inform some stage of care .

T/F

F. Every stage

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Molecular diagnostics” assess health at a ________ level, identifying gene, RNA, and protein variations that cause disease.

molecular

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A giant stride in molecular biology was ________________, a feat that took over _____ years and $______ . (The Human Genome project,_______)

sequencing the entire human genome

$2.7 billion

2003

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Remarkably, molecular diagnostics now can sequence a person’s entire genome for a cost that is rapidly declining and now approaches $____.

1000

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Molecular diagnostics is transforming health care
(1) Evolution of the field of ______
(2)_____ diagnosis
(3)______ options
(4) Pathogen ________ and ________
(5)_____ screening
(6) Drug ______
(7)____________ identification

personalized medicine

Disease; Treatment

identification and quantitation; Prenatal

discovery; genetic biomarker

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DNA is composed of two interlocking, ______ strands (the famous __—-), each of which is a string of four chemical bases in varying sequence: adenine (A), guanine (G), cytosine (C), and thymine (T).

helical

double helix

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The bases are either purines or pyrimidines. The purines are ______ and _____ and the pyrimidines are __________

adenine and guanine

cytosine, uracil and thymine

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Difference between a nucleotide and a nucleoside

A nucleotide is a nucleoside plus phosphate

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RUDIMENTS OF MOLECULAR BIOLOGY

The ______ base pairs of DNA in humans is organised into the _____ complement of __ chromosomes

The exact sequence of this _____ bases defines the uniqueness of each individual

3 x 10^9 ; haploid; 23; 3 billion

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The sequence of the nitrogenous bases is more than ____% identical in all people.

The ___ percent of sequences that are variable is responsible for the differences that occur among humans.

one

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Another function of DNA is to provide a template for transcription of approximately _____ genes that encode a variety of RNA molecules which in turn directs the production of the proteins (translation) that make up the machinery of our cells—and, consequently, the structures and functions of our bodies.

100,000

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Our _____, which are the functional units of DNA, are segments of the DNA strand that range in size from ______ bases to more than _______ bases.
The complex specialization, or differentiation, of cells throughout the body is a result of only specific genes being active in certain cells at certain times, and therefore expressing proteins related only to the functions of those cells.

genes

a few hundred

2 million

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The study of the activities and interactions of our genes is referred to as _____, and the related study of protein activity is referred to as ________.

genomics

proteomics

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A particular gene whose progression of base pairs is typical of the population at large is often termed the “ ____ type” or “____” gene sequence.

wild

normal

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A gene variant present in more than one percent of the population is called a _______ and may be considered a normal variant.

polymorphism

normal

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There are three kinds of gene mutations, which may be a change of only one base or a long sequence of bases.

_______,________,________

Hereditary, denovo, somatic

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There are three kinds of gene mutations, which may be a change of only one base or a long sequence of bases.

These are
(1) hereditary – a mutation that is passed from ______ to _____

(2) de novo –____ mutations that arise in the _________, or shortly after ______, and so are repeated throughout the body; or

(3) somatic – those that arise during ____ from _______ causes or through an error in _________, and are typically present in tumor cells.

parent to child

new; egg or sperm cell; fertilization

life; environmental; DNA replication

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Depending on the nature of the biomarker in question, molecular diagnostics therefore can assess a person’s risk of developing a disease (________), determine whether a person is a carrier of a hereditary condition (_____), screen for diseases that are present but not yet symptomatic (______), or provide a diagnosis of existing symptoms(homozygous mutation in ______ genes in cystic fibrosis).

TCF7L2

HBAS

BRCA 1

CFTR

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TECHNOLOGY OF MOLECULAR DIAGNOSTICS
In situ hybridization/ Fluorescence in situ hybridization (FISH)

A labeled DNA or RNA strand (____) is added to a _____ spread of chromosomes on a ____ which _____ with the target, complementary DNA sequence and thereby identifies and quantifies the target sequence

The exact area of hybridization is localized by layering _________ over the slide followed by ______ identification of the chromosome or by Fluorescence in situ hybridization (FISH) a (more or less?) sensitive technique

This often places the gene at a location on a given ___ or ___ on the chromosome. Both procedures have helped in Gene Mapping

probe; metaphase; glass slide; hybridizes

photographic emulsion; histologic; more

band; region

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Gene Mapping :____ Genes to ____ Chromosomes

Localizing specific

Distinct

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TECHNOLOGY OF MOLECULAR DIAGNOSTICS

CHIPS AND MICROARRAYS: The DNA microarray is a tool used to determine whether the DNA from a particular individual _______________.

contains a mutation in genes

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TECHNOLOGY OF MOLECULAR DIAGNOSTICS

CHIPS AND MICROARRAYS:

The chip consists of a (small or large?) (glass or plastic?) plate encased in (glass or plastic ?) . On the surface, each chip contains ______ of (short or long?) (synthetic or natural?) (single or double?) stranded DNA sequences which together add up to the ____________ and ——————-

small; glass;plastic

thousands of short, synthetic, single

Normal gene in question, and to variants (mutations) of that gene that have been found in the human population.

CHIPS AND MICROARRAYS
To determine whether an individual possesses a mutation for a particular disease, a scientist first obtains _______ from the patient’s blood as well as a _______
Both DNA are ________ and _____ into smaller manageable fragments and then each fragment is labelled by _________.

The individual’s DNA is labelled with _____ dye and the control - or normal - DNA is labelled with ____ dye. Both sets of labelled DNA are then _______ and allowed to _____ or ______- to the ______ on the chip.
If the individual does not have a mutation for the gene, ______________ will _____________. If the individual does possess a mutation, the individual’s DNA will __________________

a sample of DNA; normal control sample

denatured to single strands; digested; attaching a fluorescent dye

green; red; inserted into the chip; hybridize - or bind ; synthetic DNA

both the red and green samples ; bind to the sequences on the chip that represent the sequence without the mutation (the “normal” sequence)

bind to the sequence on the chip that represents the mutated DNA.

TECHNOLOGY OF MOLECULAR DIAGNOSTICS MASS SPECTROMETRY (MS): A technology that determines the _____ of a charged particle by measuring its _________. This technology is used to find and analyse ___ based biomarkers

molecular mass mass-to-charge (m/z) ratio protein

Tandem mass spectrometry has found application in new born screening for _________________.

in born errors of metabolism.

TECHNOLOGY OF MOLECULAR DIAGNOSTICS MASS SPECTROMETRY (MS): It is most generally used to find the _____ of a sample by generating a ______ representing the masses of the sample’s components. Example, because both the increase in phenylalanine and the decrease in tyrosine levels seen in phenylketonurea can be identified, the ratio of phenylalanine to tyrosine (Phe/Try) can be calculated.

composition; mass spectrum

TECHNOLOGY OF MOLECULAR DIAGNOSTICS SEQUENCING (CE, NGS): a technique used to map out the ______________ that comprise a strand of DNA. Today, this can be done via __________ or through multiple next generation sequencing (NGS) methods.

sequence of the nucleotides capillary electrophoresis (CE)

DNA Sequence Detection using Polymerase Chain Reaction (PCR) One of the essential methods underlying many molecular diagnostics is _______, the process of making copies of a specific DNA or RNA sequence found in a sample (e.g., blood or tumor) until there are so many copies that they can be detected and measured.

amplification

_____________ is the most widely used amplification technique and is considered a work horse in molecular diagnostics.

Polymerase chain reaction (PCR)

Chemist _______ received a nobel Prize for inventing PCR in 1983.

Dr. Kary Mullis

PCR is a powerful tool for locating short segments of a gene where (known or unknown?) critical mutations or variances can lead to altered cell functions associated with disease or altered function.

Known

PCR tests for the presence of a portion of DNA that has a ________, employing the same enzymatic process used by natural DNA replication to rapidly amplify, or copy, that sequence until there are thousands or millions of copies.

known base sequence

Early researchers used ________ to detect PCR products but now the use of ________, that bind to the target DNA segment as it is amplified, enable ______ instruments to monitor the resulting fluorescence without using ___. In this manner, technologies such as quantitative real- time PCR not only can ________________, but also ___________ that was originally present in the sample.

gel electrophoresis fluorescent dyes automated; gels detect the presence of the target DNA segment quantify the amount of target DNA

Most PCR tests used chemical reactions that rely on rapid cycles of temperature change to generate new strands of DNA PCR provides a sensitive, selective, and extremely rapid means of amplifying a desired sequence of DNA. Specificity is based on the use of ____________ that hybridize to ______ sequences on opposite strands of DNA and ____ the target sequence

two oligonucleotide primers complementary; flank

PCR has found application in (1) _______ medicine, (2) to detect infectious agents, especially ____ viruses; (3) to make ______ genetic diagnoses; (4) to detect __________; (5) to establish precise tissue types for _______; and (6) to study ______

forensic; latent prenatal allelic polymorphisms transplants; evolution

Screening Test Example: HPV About ____ years ago, researchers discovered a link between the human papilloma virus (HPV) and cervical cancer. It is now known that, of the 100 or so genetically distinct types of HPV, about _____ are associated with a high risk of cervical cancer, and just ____ of those are responsible for almost all cases Because HPV types are ________, it has been possible to develop molecular diagnostic tests to screen women for the high risk types

20; fourteen eight genetically identifiable

HPV The American Cancer Society recommend that women __-__ years old should receive a Pap test every ____ years, and a ___ test together with an HPV test every ____ years.

21-65 three cytological; five

Screening/Diagnostic Test Example: Cystic Fibrosis The ______ test is an example of a molecular diagnostic test that can be classified as a screening test or a diagnostic test, depending on _______.

cystic fibrosis when it is used

Cystic fibrosis (CF) is an inherited chronic disease that mainly affects the ____ and ____ and currently afflicts about _____ children and adults in the United States (____ worldwide). About ____ new cases are diagnosed each year.

lungs and digestive system 30,000; 70,000 1000

CF is caused by an abnormality in the ____________ gene that prevents the normal movement of _____ across membranes.

CF transmembrane regulator (CFTR) chloride ions

Cystic fibrosis is characterized by a build up of ___ in the lungs, pancreas, and other organs of the body. Over time, this results in patients having serious breathing and digestive difficulties and an average life expectancy that is less than ____ years.

mucus; 40

CF is seen in patients with ___ altered CFTR genes, one from each parent. Those that only have one copy of the altered gene are considered “_____”, and while they (do or do not ?) have symptoms of CF, they can ___________.

two; carriers do not pass the gene to their children.

One of the most common mutations causing CF is the __________ mutation which can be detected using a number of molecular techniques. These tests use DNA amplification methods to amplify key portions of the CFTR gene and look for the mutations that cause CF.

delta F508 deletion

CFTR gene mutation There can be prenatal ____, _____ is also done on amniocentesis samples to directly assess CF status in the unborn child. Newborns are screened in all 50 states to assess CF status. Testing can also be done on the newborn or later in childhood to ____ CF.

screening testing diagnose

Diagnostic Test Example: Chlamydia/Gonorrhea One of the most common infectious disease tests is the molecular test used to detect ________ (a CT/GC test). Because co-infection is (common or rare?) , a _____ molecular test allows for amplification and detection of both Chlamydia ______ (CT) and Neisseria gonorrhoeae (GC) DNA. These sexually transmitted diseases are caused by bacteria that can be detected through urethral or cervical samples (as well as a urine sample for gonorrhea) using molecular testing.

chlamydia and gonorrhea common combined trachomatis

CT/GC testing A (slow or rapid?) (_______) molecular CT/GC test for use at the point of care has recently been developed, providing patients with an immediate diagnosis and helping to ensure appropriate follow up.

Rapid 90mins

Therapeutic Decision-Making Example: KRAS K-Ras is a protein, encoded by the ____ gene, that plays a critical role in cell division, cell differentiation, and apopotosis. KRAS mutations that produce an abnormal, overactive K-Ras protein are found in pancreatic, colorectal, lung and other cancers.

KRAS

Detecting a KRAS mutation is used to determine patient suitability for certain therapies in ______ and _____ cancer.

colorectal and lung

Therapeutic Decision-Making Example: KRAS Anti- _____(_________) drugs (Erbitux (cetuximab) and Vectibix (panitumumab)) are key therapies for those cancers, but patients whose cancers contain KRAS typically ____ to these anti-EGFR therapies.

EGFR epidermal growth factor receptor fail to respond

KRAS mutation testing is typically performed for patients with ________ or _________ cancer via direct DNA sequencing or PCR and other amplification-based methods.

metastatic colorectal cancer or metastatic lung

Therapeutic Monitoring Example: HIV Some of the most common monitoring molecular tests are tests for ______ in patients affected with HIV.

HIV viral load

HIV viral load tests are ______-based tests that measure the amount of ______ to determine how many copies of the virus are present (measured in ________ of blood).

amplification HIV RNA copies per milliliter

Keeping ______________ is key to staying healthy with HIV.

viral levels as low as possible

The HIV viral load test is not used for _________ (this is often done with _____ tests), but typically also is used upon initial diagnosis as a _______, to get a first report on the ___________ prior to treatment.

initial diagnosis ; antibody; staging diagnostic progression of the disease

_______ is the screening test In HIV As well as ______

ELISA Western blot

PCR components Western blot- test for ________ Northern blot- test for _____ Southern blot - test for ______ South western blot- test for _____

Proteins RNA DNA DNA and binding proteins