Introduction to molecular diagnostics

Question 1

____________ 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.

Answer 1

Diagnostic tests

Question 2

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

T/F

Answer 2

F. Every stage

Question 3

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

Answer 3

molecular

Question 4

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

Answer 4

sequencing the entire human genome

10

$2.7 billion

2003

Question 5

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

Answer 5

1000

Question 6

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

Answer 6

personalized medicine

Disease; Treatment

identification and quantitation; Prenatal

discovery; genetic biomarker

Question 7

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).

Answer 7

helical

double helix

Question 8

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

Answer 8

adenine and guanine

cytosine, uracil and thymine

Question 9

Difference between a nucleotide and a nucleoside

Answer 9

A nucleotide is a nucleoside plus phosphate

Question 10

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

Answer 10

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

Question 11

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.

Answer 11

99

one

Question 12

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.

Answer 12

100,000

Question 13

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.

Answer 13

genes

a few hundred

2 million

Question 14

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 ________.

Answer 14

genomics

proteomics

Question 15

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

Answer 15

wild

normal

Question 16

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

Answer 16

polymorphism

normal

Question 17

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

_______,________,________

Answer 17

Hereditary, denovo, somatic

Question 18

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.

Answer 18

parent to child

new; egg or sperm cell; fertilization

life; environmental; DNA replication

Question 19

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).

Answer 19

TCF7L2

HBAS

BRCA 1

CFTR

Question 20

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

Answer 20

probe; metaphase; glass slide; hybridizes

photographic emulsion; histologic; more

band; region

Question 21

Gene Mapping :____ Genes to ____ Chromosomes

Answer 21

Localizing specific

Distinct

Question 22

TECHNOLOGY OF MOLECULAR DIAGNOSTICS

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

Answer 22

contains a mutation in genes

Question 23

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 ——————-

Answer 23

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.

Question 24

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 __________________

Answer 24

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.

Question 25

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

Answer 25

molecular mass

mass-to-charge (m/z) ratio

protein

Question 26

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

Answer 26

in born errors of metabolism.

Question 27

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.

Answer 27

composition; mass spectrum

Question 28

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.

Answer 28

sequence of the nucleotides

capillary electrophoresis (CE)

Question 29

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.

Answer 29

amplification

Question 30

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

Answer 30

Polymerase chain reaction (PCR)

Question 31

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

Answer 31

Dr. Kary Mullis

Question 32

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.

Answer 32

Known

Question 33

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.

Answer 33

known base sequence

Question 34

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.

Answer 34

gel electrophoresis

fluorescent dyes

automated; gels

detect the presence of the target DNA segment

quantify the amount of target DNA

Question 35

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

Answer 35

two oligonucleotide primers

complementary; flank

Question 36

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 ______

Answer 36

forensic; latent

prenatal

allelic polymorphisms

transplants; evolution

Question 37

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

Answer 37

20; fourteen

eight

genetically identifiable

Question 38

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.

Answer 38

21-65

three

cytological; five

Question 39

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 _______.

Answer 39

cystic fibrosis

when it is used

Question 40

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.

Answer 40

lungs and digestive system

30,000; 70,000

1000

Question 41

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

Answer 41

CF transmembrane regulator (CFTR)

chloride ions

Question 42

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.

Answer 42

mucus; 40

Question 43

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 ___________.

Answer 43

two; carriers

do not

pass the gene to their children.

Question 44

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.

Answer 44

delta F508 deletion

Question 45

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.

Answer 45

screening

testing

diagnose

Question 46

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.

Answer 46

chlamydia and gonorrhea

common

combined

trachomatis

Question 47

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.

Answer 47

Rapid

90mins

Question 48

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.

Answer 48

KRAS

Question 49

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

Answer 49

colorectal and lung

Question 50

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.

Answer 50

EGFR

epidermal growth factor receptor

fail to respond

Question 51

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

Answer 51

metastatic colorectal cancer or metastatic lung

Question 52

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

Answer 52

HIV viral load

Question 53

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).

Answer 53

amplification

HIV RNA

copies per milliliter

Question 54

Keeping ______________ is key to staying healthy with HIV.

Answer 54

viral levels as low as possible

Question 55

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.

Answer 55

initial diagnosis ; antibody; staging diagnostic

progression of the disease

Question 56

_______ is the screening test In HIV

As well as ______

Answer 56

ELISA

Western blot

Question 57

PCR components

Western blot- test for ________

Northern blot- test for _____

Southern blot - test for ______

South western blot- test for _____

Answer 57

Proteins

RNA

DNA

DNA and binding proteins