GM 03: Testing Flashcards by Nuna H

Genetic tests often performed on (X) material that’s purified from patient’s (Y) samples.

X = DNA 
Y = blood

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T/F: Only blood samples can be used for genetic tests.

False - some cases require other tissues to be examined

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Abnormal events during (X) can cause disruption of normal (Y). These are gross structural/numerical changes that are detected with techniques that (Z).

X = meiosis;
Y = chromosome number and structure;
Z = image whole chromosomes

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List techniques that detect abnormalities in whole chromosome number/structure.

Karyotype (and spectral karyotyping (SKY))
(Interphase) FISH
Chromosomal microarray

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Karyotype allows visualization of (X), stained with (Y).

X = metaphase chromosomes
Y = Giemsa dye

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(X) imaging technique paints each chromosome a different color. (Y) probes are used to target (Z).

X = Spectral Karyotyping (SKY)
Y = DNA
Z = specific chromosomes

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In SKY, you would expect to see (X) number of each colored (Y) in a diploid sample.

X = 2
Y = chromosome

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Name one key benefit to using SKY. Which abnormality is visually obvious?

Contrasting colors make presence of translocation obvious

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(X) chromosomal test(s) require both harvesting and amplification of patient’s cells.

X = karyotyping

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T/F: Interphase FISH technique requires neither harvesting nor amplification of patient cells.

False

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List the main characteristics that can be visualized/identified in karyotyping.

Number
Size/structure
centromere position
Banding pattern

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To avoid the need to harvest and amplify patient cells for chromosomal testing, which method(s) can be used?

(Interphase) FISH

2. Chromosomal microarray

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Interphase FISH uses probes directed toward:

Particular chromosomal region

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Interaphse FISH technique primarily used to ID changes in:

Chromosomal copy number

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How can Interphase FISH be used to ID a translocation?

Two probes from different chromosomes are found to co-localize

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T/F: Interphase FISH test results should always be verified with full karyotyping.

True

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Key disadvantage of microarray.

Only able to pinpoint changes in copy number, not spatial organization

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Microarray can’t detect (X) rearrangement/translocation due to its inability to detect (spatial/numerical) changes in chromosomes.

X = balanced;

Spatial

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PCR and (X) blotting techniques are primarily used to detect changes in (Y) of (Z).

X = Southern;
Y = size
Z = genes

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T/F: Generally, PCR will not detect subtle change in DNA sequence.

True

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How can a PCR-based approach, aka (X) approach, be modified to identify specific changes to gene sequence?

X = allele-specific PCR

The expected mutation is known

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How would you design the PCR (X) in allele-specific PCR to detect subtle change?

X = primers;

Complementary to either wild-type or mutant allele sequence

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In allele-specific PCR, what tells you the patient has mutant (X)?

X = allele;

If you use primer complementary to mutant allele and the target amplification proceeds

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Aside from allele-specific PCR, a similar method is allele-specific (X). This method uses which part of genetic information?

X = hybridization;

Full genomic DNA

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Allele-specific hybridization. (X) is hybridized to (Y). Which tool is used to then detect (Z)?

X = complete genomic DNA; Y = spot on nylon membrane Labeled oligoNT probe Z = either normal or mutant allele

T/F: Allele-specific hybridization allows you to skip electrophoresis step of allele-specific PCR.

True

T/F: DNA separated by size in allele-specific hybridization.

False

How could disease exhibiting allelic heterogeneity be detected via allele-specific PCR/hybridization?

Can't - a more unbiased approach needs to be implemented

A preferred testing method in cases of allelic heterogeneity is to directly ID (X) through (Y). What's the classical method for doing this?

``` X = the single BP sequence changes Y = DNA sequencing ``` Dideoxy (Sanger) sequencing

Sanger sequencing is a method similar to (X), using mixture of (Y).

``` X = PCR Y = dNTPs and (labeled) ddNTPs ```

T/F: Sanger sequencing allows you to skip electrophoresis step.

False

Why is Sanger sequencing a (good/poor) method for detecting mutation causing cystic fibrosis?

Poor; | If not caused by delta(F508) mutation, limited by read length (can't sequence entire CFTR gene to find mutation)

How can Sanger sequencing be improved for detecting mutation causing disease with high allelic heterogeneity (like cystic fibrosis)?

1. Sequence only the exons (harbor most predictable mutations) 2. Test for most common mutations

Detection of a "variant of unknown significance" can occur with (X) testing, such as (Y).

``` X = sequence-based; Y = Sanger sequencing method ```

What does it mean to detect variant of unknown significance?

Identifying sequence change that hasn't been previously characterized and don't have predictable effect on gene product

The two key limitations to sequence-based testing (i.e. Sanger) can be worked around via which approach?

Test affected family members first (even if not the patient)

If patient exhibits disease pathology suggestive of particular disorder, but mutations can't be ID'd by exonic sequencing methods, which test is offered next?

Linkage tests

T/F: The same types of DNA markers used in linkage analysis can also be used for linkage testing.

True

In linkage testing using RFLPs, what could you do with the patient's (mRNA/DNA)?

DNA; 1. Digest then Southern blotting 2. PCR, digest, electrophoresis

In linkage testing using SSLPs, what could you do with the patient's (mRNA/DNA)?

DNA; Just amplify (PCR) and electrophoresis

In linkage testing using SSLPs, what is the (mRNA/DNA) digested with?

No need to digest - sequence lengths vary already, so just amplify and electrophoresis

What's uniparental disomy?

Individual actually enherited both copies of chromosome from same parent

Uniparental disomy can be detected as (X) on karyotype.

X = normal

T/F: Uniparental disomy can cause unaffected parent to pass on recessive disorder.

True - if carrier

Which methods can be used to detect uniparental disomy?

Linkage testing

What's a haplotype?

Group of genes in that are inherited together from a single parent

You're use (X) testing to verify suspicions of uniparental disomy. If you're correct, you expect all (Y) to match (Z) from (one/both) parents.

X = linkage Y = marker loci Z = haplotype(s) One parent

In uniparental disomy, heterodisomy means the individual inherited (X) from (one/both) parent(s). Where did the (Y) error occur?

X = non-identical chromosomes; One Y = nondisjunction Meiosis I

In uniparental disomy, isodisomy means the individual inherited (X) from (one/both) parent(s). Where did the (Y) error occur?

X = identical chromosomes; One Y = nondisjunction Meiosis II

You're use (X) testing to verify suspicions of uniparental disomy. If marker alleles are homozygous for one parent's alleles in a haplotype, which phenomenon is seen?

X = linkage | Isodisomy

Name limitations to linkage genetic tests.

1. Require samples from multiple (heterozygous and affected) family members 2. Recombination can separate disease locus and marker

(X) testing strategy to ID uniparental disomy (can/can't) also ID imprinting defects.

X = haplotype; | Can, but only if derived from single parent via UPD or deletion

List the methods that can lead to Prader-Willi Syndrome. Star the one overlooked by haplotype testing.

1. Deletion of paternal 15q11-13 region 2. Uniparental (maternal) disomy of same region 3. Imprinting defect*

List the methods that can lead to Angelman Syndrome. Star the one overlooked by haplotype testing.

1. Deletion of maternal 15q11-13 region 2. Uniparental (paternal) disomy of same region 3. Imprinting defect*

What's a limitation to diagnostic tests (like sweat test for CF)?

1. Can't make conclusions about individuals who may not have developed disease yet 2. Can't yield insight about carrier status

When testing a minor child, if the carrier state doesn't affect his health but does affect future reproductive decisions, should carrier status be revealed to parents?

No - preferable to wait until adult years

Describe isoelectrical focusing.

Separating different molecules by differences in their isoelectric point (pI)

A biochemical testing for hemoglobinopathy analysis is (X), which is essentially protein electrophoresis that separates Hb subunits according to (Y), within (Z) gradient.

``` X = isoelectric focusing Y = size and charge properties Z = pH ```

List some situations in which profiling chromosomes may be appropriate/necessary.

1. Problems in early growth/dev 2. Infertility 3. Older-age mother 4. Neoplasia 5. Still birth 6. Family history

In chromosomal microarray, (X) is labeled green and (Y) is labeled red. What are you comparing?

``` X = normal/reference DNA Y = fetal/patient DNA ``` Compare amount of genetic info between the two (to detect extra/missing copies)

Chromosomal microarray: yellow spot indicates (X).

X = equal copy/copies between reference and fetal sample

Chromosomal microarray: red spot indicates (X) and green spot (Y).

``` X = extra copy/copies in patient sample Y = missing copy/copies in patient sample ```

Chromosomal microarray can detect (X) BP and karyotypes can detect (Y) BP.

``` X = under 0.5 million Y = 7-10 million ```

(focused/expanded) panel of mutations is better testing strategy for increasing allelic heterogeneity.

Expanded

(Gene/whole genome) sequencing is better testing strategy for increasing allelic heterogeneity.

Whole genome

(Single mutation test/panel of mutations) is better testing strategy for increasing allelic heterogeneity.

Panel of mutations

(Gene sequencing/expanded panel of mutations) is better testing strategy for increasing allelic heterogeneity.

Gene sequencing

Which technique can be | used to measure the relative expression of many gene transcripts simultaneously?

Microarray

(X) technique measures relative amounts of DNA | between two samples. This is well suited to measure (Y) in genome.

``` X = Comparative genomic hybridization Y = copy number variation ```

Why is PCR a (good/poor) technique to use to detect a gene deletion?

Poor; Better suited to identify small changes that can be contained within interval small enough for amplification by primers/Taq