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1-Molecular Diagnostics

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Swelling of fingers in baby...suspected diagnosis?
Splenomegaly
What's the normal range for female hemoglobin?
11-13 g/mL
Bony infarctions in sickle cell anemia will lead to...
Unequal finger length
Phenotypic trait characteristic of sickle cell anemia...
Abnormal beta-polypeptide in HbS
WTF does "abnormal beta-polypeptide in HbS" mean?!??!
Means a low solubility of reduced HbS (double hit)
What are the two pathways a sickling can cause?
1) Increased viscosity and clumping
2) Destruction of sickle cells
What are the disorders encountered in these two pathways?
-For Viscosity/Clumping pathway, get Ischemia, Thrombosis, or Infarction (VC ITI)
-For Destruction pathway, get Anemia (D A)
If the sickle cells aren't destroyed, why might viscosity increase?
Viscosity increases because sickle cells find it harder to move
If something is viscous, it means it has a high resistance to flow...thus, since the sickle cells find it harder to move, the resist the flow of blood
Wtf is pleiotrophy?
Refers to when a single gene influences multiple phenotypic traits
How does sickle cell anemia occur, and what does this have to do with pleiotrophy?
Via a single gene mutation from Glu to Val...this can cause multiple phenotypes
What genes are involved in hemophilia?
Factors VIII and IX (F8 and F9)
Which signaling cascade in involved in hemophilia?
Blood clotting cascade
What mode of inheritance does hemophilia portray?
X-linked recessive
Severity levels of Hemophilia A are...
Severe, Moderate, and Mild
nice..but can you put the severity levels in order from most common to lease common...
Severe (50%), Mild (40%), Moderate (10%)
Also for these severity levels...tell me the percentages of residual F8 activity
Severe will be < 1%
Mild will be 6%-30%
Moderate will be 1%-5%
The take home point is that you can have hemophilia but it varies in its phenotypic severity...some people have little or no function of F8 if the disease is severe, and some people have a moderate residual function if the disease is not so severe
How can hemophilia A be managed?
Transfusion of purified F8 (transfusion b/c too big to synthesize in labs)
What's the birth incidence of hemophilia A?
1 in 4,000 males
Where is beta-thalassemia common (geographically?)
Africa and the mediterranean
What are some symptoms of beta-thalassemia?
Hyperchroma in blood smear, many nucleated red blood cells; marrow expansion leading to osteoporotic hands and abnormalities of skull bones; iron overload in liver
What are five tests that use DNA technology?
"Surf PC" --> SRF PC
Southern blotting, RFLPs (Restriction fragment length polymorphisms), FISH (Fluorescent in situ hybridization), PCR, (Polymerase chain reaction), CGH (Comparative genomic hybridization)
What do all five of the aforementioned tests rely upon?
Nucleic Acid Hybridization
True or False: Strands with mismatches will denature at LOWER temperatures than perfectly matched strands
True. (something that is already impure is easier to degrade "further", hence requiring less temperature)
True or False: Hybridization doesn't need the strands to be COMPLETELY complimentary
True. As long as enough hydrogen bonds are made, hybridization will occur (do not need all to form)
What are the three types of nucleic acid hybrids we use in techniques?
DNA-DNA, DNA-RNA, RNA-RNA
What technique is a DNA-DNA probe used for, and what does this technique tell us?
Southern blot...discriminates on size and sequence
What technique is a DNA-RNA probe used for, and what does this technique tell us?
Northern blot...transcript size and tissue distribution
What technique is a RNA-RNA probe used for, and what does this technique tell us?
In situ hybridization...localization of expression
What does a western blot detect? With what? When?
Detects protein...with antibody...after electrophoresis & electro-transfer
What happens to DNA and probes during the southern blot process?
On a gel, target DNA is fixed, and then you let the probe run....it finds the target and anneals to it
What structure must be attached to a probe in order for it to be functional?
A label
What are two types of DNA probes...give a structural description of each
-DNA probes, which are double stranded, and .1kb to hundreds of kb for conventional DNA clones, .1kb to >20kb for PCR products

-Oligonucleotide probes, which are single stranded and 15-50 nt (usually 20-25 nt) long
Describe simply how the two probes are made
Oligonucleotide probes are made in a lab

DNA probes are clones (cell based or PCR)
What is the unique to oligonucleotide probes?
They give us great specificity
How are the probes labeled?
Oligonucleotides are labeled via end labeling

DNA probes are labeled by copying a template strand with labeled nucleotides being used for new strand synthesis
Describe random primer labeling
Soooo you denature DNA, then use a random hexanucleotide primer than can join anywhere (since they're not specific)...once the primer anneals to the DNA, you use the Klenow subunit of E.coli DNA Polymerase I (this takes care of the polymerase activity)...then add all 4 nucleotides, one of which is labelled....this allows us to sequence the DNA and check for any abnormalities (if we already know what the sequence is supposed to be)
When is random primer labeling used?
When you want to label a fragment from a gel but have no idea what it's sequence might be
What are the 5 things that affect the stability of nucleic acid hybrids? How do they increase stability?
The Nature of the nucleic acid, Length of the complimentary sequence, Temperature, [Na+], pH (basically it's all about the number of H bonds) (NLTSP = No Laws to Stop Pot)

More stability = longer...lower temp...more sodium (positive cations block repulsion of phosphate backbone)...acidic (lower) pH...more H bonds
Regarding the nature of the nucleic acid, arrange the hybrids in order of most stable to least stable
RNA-RNA > RNA-DNA > DNA-DNA
Regarding southern blotting, why do you have to already know what your sequence of interest is?
Because you have to already have a probe for it
What is southern blotting used for?
Identifying fragments by size and sequence..."southern blotting should be used to check for presence/absence of a sequence of interest or to check the size of a restriction fragment on which it is located"...needed to examine DNA fragments larger than a few kb...need to know sequence you are looking for
Describe the steps of southern blotting
Digest DNA with restriction enzyme (get diff size fragments)...initiate gel electrophoresis (fragments move from negative to positive pole...with smaller fragments moving farther and faster)...denature the DNA in alkali and transfer from gel to plastic film (nitrocellulose or nylon membrane)...where the fragment of interest is revealed by hybridization to a labeled probe (excess probe is washed off, leaving labels only where hybridization has occurred)
What else can southern blots be used for?
Can be used to discriminate the alleles of an individual...if a certain mutation changes the size of a restriction fragment...this is called restriction fragment length polymorphisms
How do you tell which individuals are heterozygous for gene deletions in a southern blot analysis?
so in southern blots we're looking at double stranded DNA...which is then denatured on the gel, giving two segments representing equal parts of a maternal and paternal chromosome...thus if see bands which are a little less dark than all the others, most likely heterozygous individual (homozygous deletions are much lighter or not even visible)
Why do we probe for COL10A1 after a southern blot
To make sure we put enough target DNA in the chambers/wells (this also shows that a lighter band is not homozygous normal (and this is not due to their not being enough DNA, since the COL10A1 shows if you have equal DNA from all patients)
in RFLPs, what is the polymorphic restriction site near to?
The mutation (holy ****, finally a short answer...oops just made it long...sorry)
What are RFLPs a subset of? Why?
They are a subset of SNPs (Single Nucleotide Polymorphisms) because an RFLP is basically just an SNP that creates or abolishes a restriction site...that's why it's called RFLP
What is the contributing reason to why the number of informative meioses might be limited?
There are only two alleles involved in an RFLP
How far can a mutation be from a restriction site
Kbs (kilobases)
What do we use RFLPs for?
To track a mutation in a family
True or False: We need to know the precise nature of the mutation (RFLP)
False, we're just tracking regions...not looking for genotypes
What does the principle of linkage have to do with RFLPs?
A variant (polymorphism) in close proximity to the mutation will be co-inherited
Where will the probe hybridize (RFLP)
To both alleles (+/- mutation)
True or False: The RFLP is present in the population at large
True
Since the mutation only arises on one allele in a particular family...what does this mean about the linkage we track?
Basically...an RFLP is a site where there's a POSSIBILITY for polymorphism, due to mutation
How does severe Hemophilia A occur?
Result of an intrachromosomal rearrangement on the F8 gene...which changes the length of the DNA between restriction sites...which changes the size of fragments on a southern blot
What are two ways to get Hemophilia A?
We can get Hemophilia A from either a point mutation (which may not be visible on the gel) or a recombination event disrupting the F8 gene
In a northern blot, what is done to the RNA prior to undergoing gel electrophoresis? How is this done?
The RNA is denatured, via denaturing gel (formaldehyde keeps it denatured)
How is RNA detected in a northern blot?
RNA species are detected based on size and sequence complimentarity...Certain RNAs are only tissue specific, and we can only get certain RNAs in certain tissues...thus we can find tissue distribution using a northern blot
What can we measure with a northern blot?
Size, sequence complimentarity, and abundance in a particular tissue type
Abundance in tissue vs localization of expression
While northern blot involves abundance in tissue...this is not the same as localization of expression, which is involved with in situ hybridization
Fill in the blank: PCR is in vitro _____________ of specific DNA sequences
Amplification (we're amplifying a particular fragment of DNA to make it more sensitive for tests)
How much DNA is required for PCR?
Only ng (nanograms = advantage = very small amount)
What are two things we can use PCR for?
Diagnostics and Forensics
True or false: PCR requires sequence information
True (needs at least some)
What does it mean when we say sensitivity is a two-edged sword?
Although there's the advantage of only needing a small sample...any contamination will also be amplified, botching the results
What are the ingredients for PCR? List the KEY ingredient first.
Thermostable Taq Polymerase, Template DNA, primers, dNTPs, buffer, Mg2+
Essentially, what is PCR?
It's in vitro cloning
What can we study with PCR? How?
Small portion (100s-1000s of bps) of the genome in isolation via amplification of oligonucleotide primers
What can PCR help analyze the effects of? Give some examples.
The effects of single-nucleotide change in detail...like RFLP, mutation detection, and sequencing...PCR can also detect size variation (range=10-100 bps) NOTE: sometimes PCR is the actual test...other times it is used to give enough DNA to run other tests...
Sickle cell anemia is an example of a disease whose mutation alters what?
Restriction enzyme sites
In sickle cell anemia, what is the restriction site that gets altered? What's its sequence? What gets altered?
Hinfl. GACTC. An A changes to a T...changing Glutamic acid to Valine
What pattern of inheritance is sickle cell anemia?
Autosomal recessive
Describe how gel blots will look for sickle cell
So since it's autosomal recessive, mutants will have to be homozygous recessive, having both alleles affected, whereas a heterozygous will only have 1 affected, and thus, 3 bands will show up. In the mutants, since both alleles are affected, both HinfI restriction sites will be gone, so get one band...
How is the HbS allele identified?
Via PCR (to amplify the RFLP and make it sensitive to digestion) & restriction digest
What does the common HFE (hemochromatosis) mutation do? What does this mean in terms of gel appearance?
Introduces an extra copy of the restriction site...regarding the gel, since we're introducing a restriction site, that means we're cutting was originally going to be a heavy fragment into two smaller fragments, so you would expect the gel column for the mutant to have a band lower than the normal...in the heterozygous, you will you a faint band where the mutant lowest band is...NOTE: the mutant has 3 bands TOTAL and the heterozygote has 4 bands TOTAL...only see 2 for the mutant and 3 for the hetero because the last (lowest) band was so light that it ran off the bottom of the gel
True or False: Most single base changes DO NOT affect restriction sites
True (but for HFE, it is due to a single base change, causing the addition of a restriction site)
So can you still detect single base changes in the sequence, even when a restriction site is not affected? Is this even possible? How?
Yes, can use oligonucleotide labeled via end labeling for even when a restriction site has not been affected...
What does end labeling do?
Adds a radioactive phosphate to the 5' OH group
Oligonucleotides (synthetic DNA) are used in what 3 things?
Used as primers in PCR, probes in ASO, and linkers in library construction
What's the range on oligonucleotides...are they short or long?
Short...17-25 nt in length
What does ASO stand for?
Allele Specific Oligonucleotide
What does the specificity of ASOs depend on?
The percentage of matched H bonds
We know that oligonucelotide probes are much shorter than DNA probes...what does this mean in terms of their difference in nucleic acid hybridization?
While DNA probes can STAY hybridized and stable with a single mismatch (even up to 20% mismatch)...an oligo probe will be completely thrown off by even a single mismatch
So what is the advantage of oligo probes over DNA probes?
Since a single mismatch will throw off an oligo...this allows ASOs to detect SINGLE BASE CHANGES without the presence of an RFLP
The single base change of A to T (Glu to Val), causing sickle cell anemia...occurs in which gene?
The beta-globin gene
What kind of testing is available for sickle cell anemia?
Straightforward, specific testing (dot blot)
How does a dot blot work?
Two allele specific oligos are used to distinguish between the A (normal) allele and T (mutant for sickle) allele...both chrom (mom and dad) are tested...so you take known samples (AA and TT oligos) and put them on the same blot with the test sample in the center...then you can analyze the results of the dot blot...if there's only a dot in the A probe row, then the person is AA (normal), if there's only a dot in the T probe row, then the person is TT (sickle cell anemia), and if there are two dots, one for the A row and one for the T row, the person is AT (heterozygous=sickle cell trait)
What's the general process involved in using ASOs?
So the region of genome (usually a single exon) containing the known mutation is PCR amplified, denatured, and applied to a nylon membrane, half of which is hybridized to a normal probe, and the other half hybridized to the mutant probe
What do ASOs discriminate between?
Sequences differing by a single base
True or False: ASOs don't REQUIRE that the mutation be known
FALSE: ASOs DO REQUIRE that the mutation be known
True or False: ASOs can automated
True
What are ASOs suitable for?
Screening for frequent mutations like CF, beta-thalassemia, and Ashjenazi mutations
Summary point*****What are we doing when we use restriction enzyme digestion of DNA (amplified via PCR)
Looking for a mutation that creates or abolishes a restriction site, which would shorten or lengthen the DNA fragment of interest, respectively
Summary point****What do we do for specified point mutations?
We would PCR amplify out DNA sequence of interest and then allow it to hybridize to ASOs on either a dot blot, gene chip, or ASOs on a DNA-chip (for larger arrays to globally scan for a mutation)
What kind of diseases do we check the size of an expanded repeat for? What do larger expansions require? What about smaller ones?
For dynamic repeat diseases. Larger expansions require southern blots. Smaller expansions require PCR
What is FISH used for, and name some diseases involving this.
Large deletions (larger than southern blots, which detect deletions/rearrangements in 10s of kbs)...WDP = Williams syndrome, DiGeorge syndrome, and Prader-Willi/Anglemann
What kind of probes are used for FISH and how big should they big in comparison to the deletion?
BAC (bacterial artificial chromosome) which are 100-200 kbs...the PROBE MUST BE SMALLER THAN THE DELETION...think about it...if the probe was larger than the deletion, it would still hybridize...we want out probe to not attach if there's a mutation
Standard FISH vs chromosome painting FISH?
Standard uses a probe for a particular deletion sequence in a chromosome, while painting uses a whole array of chromosomes to look for different things like translocations (i.e. you "paint" the whole chrom instead of marking individual sections)
How does FISH work in general?
Purified clone is complimentary to the sequence that may be deleted...so you insert nucleotides attached to a fluorophore into your probe, then denature it so that it can anneal to the in situ DNA from the chromosome of interest
How many probes do you use with FISH? Why?
Two. One serves as a marker to indicate the chromosome you're looking for. The other is specific for the area of deletion...both markers should light up, and if there is a deletion in one of the chromosomes, only that chromosome with the deletion will have the region specific probe light up
What are other names for the 22q11 deletion syndrome?
DiGeorge syndrome or Velo-Cardio-Facial syndrome
What are some abnormalities associated with the 22q11 syndrome?
Short stature, cleft palate, prominent nose, flat maxillary area, mild retardation, tetrology (in heart), malformed kidneys...side note: 22q11 is a deletion of about 3 million bps
Give an example of when chromosome paining may be useful
In the diagnosis of trisomy 21...if you see hypotonia, excess nucal skin, and bilateral single palmer creases, you can use chromosome painting...let's say you mark chromosome 21 with red...so if you see three red spots, you can conclude trisomy 21
What else can we use chromosome painting for?
To reveal reciprocal translocations
What is CGH used for? When is it better than using FISH?
CGH is used to identify deletions/duplications...CGH is better to use when you don't know where the deletion/duplication is
How does CGH work?
Let's think about the name first...COMPARITIVE...GENOMIC....HYBRIDIZATION...we will be comparing cells from a patient to a normal control...so you take the cells...extract the DNA...label with two different fluorochromes...mix the two types of cells in EQUAL quantities, and place in chambers on a DNA chip...chambers which have BAC probes
How do you interpret CGH results?
So let's say the patient DNA is green and the the control DNA is red. If there the patient is normal, there will be equal amounts of red and green DNA in the chambers...thus all the chambers will show yellow...now if you see red and yellow slots, or more red slots than green slots, than you know there's more control DNA than patient DNA, which would mean a deletion in the patient....if you see green and yellow slots, or more green slots than red slots, than you know there's more patient DNA than control DNA, which would mean a duplication in the patient
How is CGH interpreted graphically?
So you will see a bunch of dots, but if you see a particular group of dots that is higher on the graph (relative to all the other dots), that means the ratio is above the normal limit, indicating a duplication...if you see a group of dots that is lower on the graph, that means the ratio is below the normal limit, indicating a deletion
In a 45X karyotyped patient (Turner syndrome), what are many of the cases due to?
Loss of chromosome after fertilization...so there is a risk of small population of 46XY cells in the gonads
What technique do we use to rule out the presence of Y specific sequences in Turner syndrome? Why do we use this over karyotyping?
We PCR for Y-specific sequences...this increases sensitivity since there may only be a small quantity of cells...karyotyping may not be able to pick up the abnormality
Why is DNA sequencing often referred to as? Why?
The GOLD STANDARD for mutation identification...it should be able to identify ANY single base substitution
What are some things sequencing is necessary for?
Many autosomal dominant conditions (like Marfan syndrome or collagenopathies) and recessive conditions that don't have a common mutation (like atypical CF and inborn errors)
What is crucial for sequencing and what is special about these?
ddNTPs (dideoxynuelcotides)...in which a lack of a 3' OH prevents chain extension
What is the principle of sequencing?
Each time a nucleotide is added into the growing chain, there is a small chance that it will be a ddNTP, which will terminate growth....so you get a series of nested fragments...electrophoresis separates the fragments by length, and each ddNTP carries a differently colored fluorescent dye
What does the importance of template quality have to do with sequencing?
Sequencing requires high quality DNA...if it isn't carefully purified, results may not be interpretable
What type of inheritance does NPS (Nail Patella Syndrome) have?
Autosomal Dominant (thus most likely found using sequencing)
What genetic makeup are patients with NPS (homozygous, heterozygous, etc)?
Heterozygous
When you have a heterozygous patient with an AD disease, how many bands would you expect to see at the same position
2. One is from mom and one of from dad....one of them has the mutation
Describe NPS.
So one chrom from either parent has the mutation...two different mutations can cause NPS...one is a point mutation of G to A causing Arg to change to Gln...The other is a point mutation of C to T causing Arg to change to Ter (termination)...and the reason you see 2 bands in the same position is because one is from normal parent (with either Normal G or C) and other is from affected or carrier patient (with G changed to A or C changed to T)....remember NPS = "GACT" sounds like "jacked"
What are other names for the base changes that occur in NPS
The G to A is R200Q (which involves loss of MspI site) and the C to T is R198X
Describe fluorescent sequencing and what does it mean if there are two peaks in the same spot for a different base?
Different colored dyes are attached to each ddNTP and the sequence is read by a computer...like in a gel two positions at the same position indicate a heterozygote
What causes beta-thallassemia?
A beta-globin mutation (specifically due to NULL mutations in the beta-globin gene)
What does a null mutation mean?
Transcription or translation will not occur (either absence of gene product or absence of any function at phenotypic level
Describe beta-thallassemia
Since intronic DNA is mutated, part of the intron gets incorporated into the exon, making it a null mutation of the beta-globin gene because it can't get translated then
How does LHON occur and what is a symptom of it?
A single base change from G to A in mitochondrial DNA...progressive worsening of central vision (called Leber's Hereditary OPTIC Neuropathy)
How can the mutation in LHON be detected?
Using restriciton enzyme digestion of PCR products...this can be used because the mutation introduces a restriction site (MaeIII) and takes out the other restriction site (SfaNI)
What does SSCP stand for what does it rely on?
Single-Strand Conformational Polymorphism...it relies on the breakdown of homologous base pairing
What is SSCP used?
When the mutation in to the gene to be analyzed is unknown
What does the analysis of single stranded DNA depend on?
The tendency of single-strand DNA to form intramolecular H bonding (hairpin loops)
What two things does the electrophoretic mobility of a DNA molecule in a non-denaturing gel dependent on?
It's conformation and length
Describe the process of SSCP
DNA is amplified by PCR...denatured...IMMEDIATELY applied to a nondenaturing gel so that renaturing with the complimentary strand does not occur and the single stranded DNA molecules remain separated from each other and begin to migrate in the gel...during which time they will hybridize with themselves by forming hairpins
In SSCP, why do the single strands migrate at different speeds?
Because of secondary structure (intramolecular H bonding)
True or False: A SINGLE base change CAN have the ability to change the secondary folding structure of a single strand of DNA
True
What is CYP2D6 an isoform of? What does CYP2D6 do? What has the mutation of this enzyme been associated with?
Cytochrome P450. Metabolizes drugs. Cancer due to altered metabolism
What does the banding pattern look like in SSCP?
Every chromosome in SSCP is going to have 2 bands...so homozygotes will have 2 bands and heterozygotes will have 4 bands
True or False: SSCP does not identify mutations in CFTR
False...it does
Why would you PCR amplify exons in SSCP?
So in SSCP you PCR amplify a bunch of different exons and look for SSCPs...whereever you find them, then THAT is the exon which you're going to sequence to find out exactly what the abnormality is
What are 3 advantages of SSCP?
1) Can screen multiple exons from multiple samples at once
2) Moderately sensitive
3) Lower cost
What are 3 disadvantages of SSCP?
1) NOT ALL POINT MUTATIONS AFFECT SECONDARY STRUCTURE
2) There is preferential identification of small insertions/deletions
3) Still have to sequence aberrant products
What makes SSCP obsolete?
Sequencing is cheaper than ever these days
What does SNP stand for and give a subset of it
Single Nucleotide Polymorphisms....RFLPs are a sub-type
What do we use DNA polymorphisms for?
We use these changes in DNA from person to person in order to map genes
What type of polymorphism is the most common in the human genome? Which provides the most information?
SNP. CA rpts (microsatellites)
How do we use VNTRs?
A type of VNTR is a (CA)n repeat...these are present in everyone at the same chromosomal locations...but the number of repeats of the dinucleotide sequence in highly variable from person to person...there is the advantage of having many possible alleles (meaning that each person can have 5,6,7,etc repeats), increasing the likelihood of informative meiosis
How do we use SNPs?
SNPs are less informative than VNTRs b/c there are usually two alternative nucleotides at a polymorphic site...BUT they are abundant and can be typed without using gel electrophoresisT
True or False: CA repeats can be used for typing
True...so you can use CA repeats/VNTRs/Microsatellites (all the same ***, and the most informative, ***)...amplify it with PCR and type people for this genetic marker to see which alleles they&#039;re carrying for it
True or False: Fluorescent labeling replaces radioactivity
True
What do we mean when we say that CA-rpt markers are like regular M&M's?
Multiple alleles, more likely that any two alleles are different...that the individual is heterozygous...like 5 colors...chance any individual will be homozygous is 1 in 25
What do we mean when we say that RFLPs are like Halloween M&M's?
Only 2 alleles...less likely that the individual is heterozygous...like 2 colors..chance any individual will be homozygous is 1 in 4
What conclusion can be drawn from the M&M's example?
CA rpt markers are much more useful in telling us which chromosome is which
How does gene tracking work?
Not diagnosing anything...just following chromosomes that carry the mutation and typing based on the chromosome inheritance...but gene typing doesn't lead to 100% possibility-can't say for sure since haven't examined the gene...
How does gene tracking work in layman's terms?
Once research has found a reliable marker that co-segregates with the disease allele, we can use gene tracking to predict the genetic risk of others in the family
I SAID LAYMAN'S TERMS DAMNIT! wtf does gene tracking mean...
BASICALLY...we're following around the diseased allele and seeing who inherited it or not via the use of markers
What are the 4 phases of genetic tracking?
1) Establish mode of inheritance
2) Informative? (who started the passing down of the asshole allele?)
3) Establish phase (which allele is the asshole?)
4) Type consultant (predict asshole's risk to reappear)
So how do you know what test to use?
Size matters...(indeed)...if there's big changes in DNA sequence, use a big probe (really?)...for deletion of 100s to 1000s of Kbs, use FISH...if changing a single nucleotide, use ASO...a probe has to discriminate between sequences...ASO is for 1bp difference (sickle), Southern is for between related genes (also works for deletions of 10s of kbs)...and BAC is for between chromosomal regions
True or False: Individuals with the same marker genotype will have the same phenotype
FALSE: There is NO REASON why individuals with the SAME MARKER GENOTYPE have to have the same PHENOTYPE