ALL THE THINGS Flashcards by Mujhtuba Baksh

What is a probe?

nucleic acid fragment of known sequence complimentary to the sequence to be detected; Often labeled

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What is the target in hybridization assays?

complex mixture of nucleic acid fragments being tested

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What is Hybridization Stringency?

degree to which non complementary sequences are tolerated during hybridization

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The higher the stringency, the fewer what?

mismatches will be tolerated

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What applications would require high stringency?

detecting a point mutation; need a perfect match

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What applications require low stringency?

identifying multiple genes in a gene family; mismatches are tolerated

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What 3 hybridization conditions determine stringency?

temperature, salt concentration, denaturing agents

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Stringency increases with what?

high temperature
high concentration of denaturing agents
low salt concentration

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What method would we use if we had a DNA probe and a DNA target?

Southern Blot - discriminate fragments based on size and sequence

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What method would we use if we had a DNA probe and a RNA target?

Northern Blot - discriminate between mRNA transcripts on the basis of sequence and size

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What method would we use if we had a RNA probe and a RNA target?

in-situ hybridization (ISH) - localization of expression of specific mRNA at the tissue and cellular level

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What is a a Western Blot used for?

detection of protein with antibody after electrophoresis and electro-transfer

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In southern blotting, when detecting DNA bands, what does the intensity of a band reflect?

the amount of target DNA present

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Would we expect to see differences between tissues in southern blots from a single patient?

No - since genomic DNA is used in southern blotting, and all a person’s cells carry genomic DNA, we wouldn’t expect to see differences

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Can we use southern blots to discriminate between alleles carried by an individual?

Yes; if a mutation/polymorphism changes the size of the DNA fragments.

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What is a Restriction Fragment Length Polymorphism (RFLP)?

a polymorphism that results in a change in the size of a restriction fragment (number of base pairs between 2 restriction sites)

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Besides a single nucleotide polymorphism, what else can cause restriction fragment sizes to change?

chromosomal rearrangement
insertion/deletion between sites

This is how other RFLPs are created

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The patient with hemophilia presented with what abnormality in the periosteum of his skull?

cephalohematoma (possibly a giveaway for questions)

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In general, what is the mode of inheritance for Hemophilia?

X-Linked Recessive

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What are the 2 types of Hemophilia?

Hemophilia A

Hemophilia B

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Where is the defect in Hemophilia A?

in Factor VIII (F8 gene)

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Where is the defect in Hemophilia B?

in Factor IX (F9 gene)

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What is hemophilia?

coagulation disorder associated with prolonged bleeding times, hemorrhage into joints and muscles

variable severity

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40% of severe Hemo A cases are caused by what?

an inversion due to homologous recombination between repetitive elements proximal to the F8 gene

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What affect does the specific inversion have on the F8 gene in Hemo A cases?

it changes the distances between restriction sites, changing the sizes of fragments observed on Southern Blot

Would a standard PCR test detect the inversions seen in Hemo A cases?

No because all the exons remain present

In the milder form of Hemo A, how is protein function affected?

we still see residual protein function

In the more severe form of Hemo A, how is protein function affected?

we see little or no protein function

How is Hemo A treated?

by transfusion of purified/recombinant F8

In Southern Blotting, when analyzing an individual heterozygous for a deletion, is there less or more DNA available to which the probe can hybridize? How does this effect the analysis?

Less - signal will be weaker corresponding to the regions deleted on the mutant. (Signal compared to that of a normal chromosome)

What do we use RFLPs for?

to track a mutation in a family

For RFLP analysis do we need to know the precise nature of the mutation?

Nope

We use the principle of linkage in RFLP analysis. What does this mean:?

that a variant in close proximity to the mutation will be co-inherited because they are on the same chromosome

In RFLP anaylsis, will the probe hybridize to both alleles?

Yep

The RFLP used for a particular RFLP analysis is present in the population at large. T/F statement?

True statement

The linkage we track in RFLP is specific to what?

it is family specific because the mutation has arisen in one allele in a particular family

What is Allele Specific Oligonucleotide (ASO) Hybridization?

hybridization based method to detect sequence variants (mutations or polymorphisms)

Do we need restriction sites in ASO hybridization?

Nope

We use ASO hybridization to discriminate between sequences differing by what/?

differing by a single base

For us to use ASO hybridization, does the mutation in question have to be known?

Yes

If we want to screen for frequent mutations, would we want to use ASO?

You bet.

What kind of probe is used in ASO hybridization?

Oligonucleotide rich probe

How many probes do we typically use in ASO hybridization?

one for each sequence variant being tested i.e. one for the wild type and one for each mutant

The site where the mutation occurs is typically centered within the oligonucleotide in ASO. Why?

to cause maximum disruption to hybridization if there is a mismatch

In ASO hybridization, our target DNA is amplified via PCR from genomic DNA. By what method is it applied to a membrane for analysis?

Dot blot

Can we separate sample based on size in ASO hybridization?

Nope because there is no gel-electrophoresis used in this method Trying to determine if the sequence is present or not

Via the Dot Blot method, the target DNA in ASO is applied to the membrane in duplicate. True or False?

True

When analyzing probes in ASO, how does a positive signal manifest itself?

it appears a black spot indicating that hybridization has occurred

What does a negative signal in ASO appear as?

it is blank; indicates the probe did not hybridize so the patient's genomic DNA doesn't have the complimentary sequence

Do we use short or long probes in ASO?

short

Why do we use short probes in ASO? 3 reasons.

want the probes to hybridize perfectly to complementary sequences, but not with sequences with single base changes Percentage of mismatched H-Bonding plays a role in how well a probe binds a mismatched sequence Short probes are more specific

With a single-base mismatch, a short probe has a greater % of mismatched H-bonds than long probes. How is hybridization affected if we use long probes instead?

hybridization is more likely to be disrupted

What disease would we want to use ASO for?

Sickle Cell Disease

What is the mode of inheritance for Sickle Cell Disease?

Auto Recessive

The mutation involved in SCD changes Adenine to what?

Thymine

The mutation in SCD results in a change from Beta-A Globin to?

Beta-S Globin (Affecteds are homozygous for this)

What is the specific mutation in SCD?

Mutation is E6V - In the 6th codon, a switch from A to T changes a Glutamate to Valine

What is PCR?

Polymerase Chain Reaction exponential in vitro amplification of specific DNA sequences

In PCR, how is DNA synthesized? Asking for the directionality of the overall synthesis.

cyclical synthesis of DNA via DNA polymerase

Describe the sensitivity of PCR?

it is a highly sensitive method

As a result of its highly sensitive nature, PCR is susceptible to what?

contamination

What is required in PCR in terms of the sequence we want to analyze?

We need some information about it. Can't just use PCR without any information

In PCR, DNA is synthesized by a what kind of polymerase? Hint, think about the environment the polymerase would be active in in this method.

a thermostable DNA polymerase

What are the 2 common polymerases used in PCR? Which is more common?

Taq (most common) | Pfu

Which PCR polymerase can proofread?

Pfu Taq cannot.

The PCR reaction occurs in repeated cycles. How does the amount of DNA product change in each cycle?

Doubles after each cycle

What are the 3 steps in PCR that are repeated over and over?

(1) Heat to separate DNA strands (2) Cool to allow primers to bind (3) DNA polymerase extends the 3' end of each primer

What are the 5 main components of a PCR reaction?

``` Target DNA 2 primers All 4 dNTPs Buffer, Mg2+ Thermostable DNA Poly ```

The progress through a PCR reaction is controlled by temperature. How is this automated?

by using thermocyclers

The region to be amplified by using Taq in PCR can be up to how many bp?

10,000 bp | double with Pfu

Each of the two primers used in PCR primes the synthesis of a different strand across the region of interest. True or False?

True

Typically, how long should PCR primers be?

20 nucleotides

Do we need to provide the sequence for synthesis of primers used in PCR?

Yes

How do we determine the sequence of the primers from the sequence of the target DNA?

One of the primers (Forward primer) is identical to the 5' end of the region to be amplified of the given strand. The other (Reverse Primer) is complementary to the 3'end of the given strand

What are the major applications of PCR?

``` DNA sequencing Genotyping Diagnostics Forensics Quantifying Gene Expression ```

Can you make cDNA and Genomic DNA clones via PCR? If so, how?

Yep. Just follow the same steps.

What is Reverse Transcriptase PCR (RT-PCR) used for?

amplification of RNA by PCR used to detect RNA viruses amplify mRNA of expressed genes

Can you use PCR to measure microsatellites?

Yep

In molecular diagnostics, restriction digestion of PCR-amplified DNA can be used only when?

when the mutation creates or abolishes a natural restriction site or if one is created by use of special primers

Hybridizing PCR-amplified DNA to allele-specific oligonucleotides on a dot-blot or gene chip is the general method for what?

method for analyzing specified point mutations

PCR using allele-specific primers (ARMS test) is a general method for what?

method for analyzing point mutations

PCR can be used to check the size of an expanded repeat. When is this useful in diagnostics?

when looking at dynamic repeat diseases only

What are some common clinical presentations of Sickle Cell Disease?

``` presents within first 2 years hemolysis hemolytic anemia splenomegaly repeated infections painful swelling of hands/feet (occlusion of capillaries) death by infection common ```

How can you treat SCD?

supportive methods including O2, transfusion, penicillin

Individuals who are heterozygotes for SCD (carry Sickle Cell Trait) are generally healthy, but may present with symptoms when?

when under stressful conditions

What is Pleiotropy? What disease in class is it associated with?

a single gene affects multiple, seemingly unrelated phenotypic traits Sickle Cell Anemia

The Sickle Cell mutation alters what kind of site?

a specific restriction site