Research Methodologies

What is the overall focus of this lecture

research methodologies and techniques that are useful for clinical diagnosis

DNA/RNA techniques

genetic testing

gene therapy

protein based techniques

medical imaging

What is the major goal of using biotechnology in clinical diagnostics

to detect, characterize, or monitor disease using biological information such as DNA, DNA, proteins, metabolites and tissue chracteristics

What are 2 examples of clinical applications

• newborn blood screening

• alzheimer’s disease screening

What type of technique is specifically associated with new born blood screening

mass spec

Why is molecular diagnostic testing useful in medicine

it can identify molecular abnormalities associated with disease and help with diagnosis, prognosis, treatment selection and monitoring

What is a SNP

a single nucleotide polymorphism

a variation at a single nucleotide position in the genome

What is a structural variation (SV)

Larger-scale variation in the genome involving changes in the structure or organization of genomic DNA

Why can differences in the noncoding regions of DNA matter

noncoding regions can contain regulatory elements that can affect gene expression

What type of regions can regulate a protein-coding gene

regulatory elements such as promoters and other noncoding regulatory sequences

What is the basic difference between a SNP and structural variation

a SNP involves a single nucleotide whereas structural variation involves larger genomic changes

Why are genetic differences important clincically

they can contribute to differences in disease susceptibility and response to treatment

What does PCR stand for

polymerase chain reaction

What is the basic purpose of PCR

to amplify a specific DNA sequence

Why is amplification important in molecular diagnostics

a patients sample may contain only a small amount of the DNA sequence of interest, so PCR creates many copies that can be detected

What major components are required for PCR

DNA template

primers

nucleotides

DNA polymerase

appropriate reaction buffer

What is the DNA sample in PCR used for

provides the template DNA that contains the sequence being amplified

What are primers in PCR

short DNA sequences that bind to the target DNA and define the region that will be amplified

What do nucleotides provide during PCR

The building blocks needed to synthesize new DNA strands

What enzyme is specifically shown as being used in PCR

Taq polymerase

Why is Taq polymerase useful for PCR

It can function at the high temperatures used during repeated PCR cycles

What instrument repeatedly changes temperatures during PCR

a thermal cycler

What are the 3 major steps of one PCR cycle

denaturation → annealing → extension

What happens during PCR denaturation

the double-stranded DNA separates into individual strands

What happens during PCR annealing

Primers bind to their complementary sequences on the DNA template

What happens during PCR extension

DNA polymerase extends the primers and synthesizes new DNA

Why does PCR produce exponential amplification

The DNA produced in one cycle becomes template for subsequent cycles, causing the amount of target DNA to increase rapidly

What is the difference between exponential and linear amplification

PCR can generate exponential amplification because newly synthesized DNA becomes template, whereas linear amplification would only ass a similar amount of product per cycle

What can be used to visualize PCR products after amplification

Gel electrophoresis

What does gel electrophoresis allow researchers to determine about DNA

it allows DNA fragments to be seperatedd and visualized based on their size

A patient has a suspected mutation in a particular DNA sequence. What technique could amplify that region for analysis

PCR

Is a diagnostic test needs to distinguish 2 different alleles what specialized PCR technique can be used

allele-specific PCR

What is allele-specific PCR

a PCR method designed to distinguish between specific alleles such as a normal allele and a mutant allele

How many PCR reactions are described for allele-specific PCR

Two PCR reactions are performed simultaneously

What is different between the two allele-specific PCR reactions

They use different primers designed to recongize either the normal sequence of mutant sequence

What is the purpose of the allele specific primer

it preferentially amplifies DNA containing the sequence that matches that primer

How can the result of allele-specific PCR be detected

By examining the resulting amplification, including through gel electrophorisis

A patients DNA produces amplification only with the mutant-specific primer

what does this suggest

the patients DNA contains the sequence recognized by the mutant specific primer

Why is allele-specific PCR useful clinically

it can provide a practical method for determining whether a patient carries a particular known sequence variant

What does GWAS stand for

genome-wide association study

what is the basic purpose of a GWAS

to identify SNPs or patterns of SNPs associated with a disease or trait

Does a GWAS usually examine one gene or the genome broadly

it examines genetic variation across the genome

what does a GWAS compare

genetic variation in people with a particular condition versus appropriate controls without the condition

What type of genetic differences can GWAS identify

differences in the frequency of particular SNPs or SNP patterns

What does altered gene frequency mean

A particular allele occurs at a different frequency in one group compared to another

What disease is used as an examples of an early successful GWAS in this lecture

Age-related macular degeneration (AMD)

What dies the AMD GWAS identify

Two SNPs that were more common in individuals with AMD than in healthy controls

What other conditions are listed as examples of diseases studied using GWAS

type 2 diabetes

alzheimer’s disease

parkinson’s disease

crohn’s disease

can GWAS also investigate treatment response

Yest

the lecture lists response to antidepressent medications as an application

Can GWAS be used to study obesity

yes

What is the difference between association and causation in GWAS

A GWAS identifies statistical association between a genetic variant and a trait; it does not by itself prove that the variant directly causes the trait

In a GWAS, why are controls important

They provide a comparison group for determining whether particular variants occur at different frequencies in people with the disease

A SNP occurs much more frequently in patients with disease X than controls

what has the GWAS demonstrated

an association between the SNP and disease X

What is another name for a gene chip

a gene array or DNA microarray

What is physically attached to the spots on a gene array

single-stranded DNA sequences with known specific sequences

What is the purpose of having many different DNA sequences on one array

to allow many different sequences to be tested simultaneously

Approximately how many spots can a single DNA chip contain

many thousands

What principle allows a sample to interact with the SNA sequences on a microarray

DNA hybridization

What does hybridization mean

complementary nucleic acid sequences bind to one another

Why can hybridization be highly specific

conditions can be optimized so that sequences preferentially hybridize when they are perfect matches

What is one way DNA arrays can detect SNPs

Patient DNA can hybrizie to array sequences designed to distingush different nucleotide varients

What is another major application of DNA arrays

Gene expression profiling

What is gene expression profiling used to compare

The activity/expression of genes between different samples such as normal vs diseased tissue

Why would researchers compare gene expression between normal and diseased tissues

to identify genes whose expression differs between the 2 conditions

What could it mean if a gene is active only in diseased tissue

it may be potentially important disease-associated marker or target

What could it mean if a gene is active only in normal tissue

it may be associated with normal cellular function and may be altered or lost during disease

What happens if a gene is active in both normal and diseased samples

its expression is not uniquely associated with the disease in that comparision

What happens if a gene is inactive in both samples

it does not distinguish the normal and diseased samples in that experiment

What molecule is extracted when performing gene expression profiling

mRNA

Why is mRNA useful for determining gene expression

mRNA reflects which genes are being actively transcribed

What is mRNA converted into for use on the DNA array in the lecture

cDNA

What does cDNA stand for

Complementary DNA

Why is cDNA generated from mRNA

It provides a DNA representation of the expressed RNA sequences that can be hybridized to the DNA array

In the example comparing tumor to normal tissue why are different fluorescent labels used

to distinguish the material origination from the different samples when both are applied to the array

What does increased fluorescence from one sample indicate on a gene expression array

greater representation/expression of the corresponding gene sequence in that sample relative to the comparison

What is one research application of gene expression profiling

identifying disease marker genes

How can gene expression profiling help cancer research

it can help identify genes involved in tumor biology

How can gene expression profiling potentially affect treatment

it can help predict tumor behavior and improve patient treatment decisions

What is personalized therapy

treatment tailored to characteristics of an individual patient or tumor

Why can two patients with the same disease respond differently to the same drug

Genetic differences can affect drug targets, drug metabolism, and other aspects of treatment response

What are three reasons mentioned for differences in treatment response

genetic differences, differences in drug activity against the target and differences in drug metabolism

What does pharmacogenetics study

how genetic variation affects drug response

What does pharmacogenetics broadly involve

using genomic information to understand disease and optimize therapy

What is the overall goal of using genetic information in personalized therapy

to improve the efficacy and safety of treatment

What CYP genes are listed in the lecture as potentially relevant to personalized therapy?

CYP2D6, CYP2C9, CYP2C19, CYP3A4/3A5, and CYP2B6.

Why are CYP polymorphisms clinically relevant

genetic variation in CYP enzymes can affect drug metabolism

What is TPMT

a gene/protein whose polymorphisms can influence drug metabolism and treatment response

What is DPYD

A gene involved in drug metabolism whose polymorphisms can be clinically relevant

What is BCR-ABL

A molecular abnormality involving the BCR-ABL fusion that cen be detected and quantified for certain cancers

What other tumor-associated alterations were discussed

• KIT/PDFFRA mutations

• HER2 amplification

• KRAS mutations

• EGFR mutations

• EML4-ALK translocations

• BRAF V600E

Why would identifying a mutation in a tumor be useful

it may help characterize the tumor and guide targeted treatment monitoring

What is gene therapy

A therapeutic approach that introduces genetic material into cells to alter cellular function or restore production of a desired protein

What are the prerequisites for the gene therapy concept described in the lecture

a single identified gene cause

a mutation that prevents normal gene function

loss/cessation of gene protein expression

replacement gene capable or producing the protein

Why is identifying a single causative gene useful for gene therapy

it provides a specific gene target that can potentially be replaced or corrected

What must happen to the replacement gene for successful gene therapy

must produce the desired functional protein

What is the basic idea behind ex vivo gene therapy

Cells are removed from the patient, genetically modified in the laboratory, and then returned to the patient

What is the major step in the gene therapy process shown in the lecture

cells are removed from the patient

What happens to the virus used for gene therapy

it is altered so it cannot reproduce

Why is a virus used in many gene therapy approaches

Viruses natually have highly efficient mechanisms for delivering genetic material into cells

What happens when the altered viral vector is mixed with patient cells

the cells can become genetically altered by reciving the theraputic genetic material

What happens after the patient cells are genetically altered

they can produce the desired protein or hormone