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Sickle cell trait

What kind of genotype does it have?
What base change causes it?
What does the mutant gene cause?



Glutamine acid--> valine

mutant gene causes abnormal β polypeptide



multiple affects from one mutation
ie: sickle cell
mutant gene-->abnormal β polypeptide in Hb-S--> low solubility of reduced Hb-S---Sickling--> 2 paths
1. increase in viscosity + clumping-->ischemia
2.destruction of sickle cells-->anaemia


Defect differences in type A vs. type B

Inheritance pattern for both?

what determines the severity of hemophilia A?

Defect in genes for:
-factor VIII for type A
-Factor IX for type B

Both are X-linked

Hemophilia A can be severe or moderate depending on the residual F8 activity

Nucleic acid hybrids

Southern blot
Northern Blot
In situ hybridization
Western blot

Which nucleic acid is used in each?
what does each measure?

-discrimination of size and sequence

-transcript size and tissue distribution

-localization of expression

W-Protein w/ antibody
-protein presence and size


Mechanism of Nucleic acid hybridization

what are the 3 possible results?

Target DNA is denatured
probe complementary to DNA is created
DNA and probe anneal
result is 3 products:
DNA anneals with itself
DNA anneals with probe (what you want)
probe anneals with self

Probe labeling

DNA vs oligonucleotides
-Strand difference?
-what sizes are used for each?

DNA- usually 2x stranded for sizes of 1kb-100s of kbs

Oligonucleotides- usually 1x stranded
-20-25nt long

Factors that affect stability of DNA hybrids (4)

increase # of H bonds-->increase stability

longer probes--> more stable

decrease temp--> increase stability

Increase pH--> increase stability

*basically comes down to the # of hydrogen bonds

Southern Blot mechanism

How does this test discriminate between fragments?
What does the mutation do to the DNA fragment?

DNA is cut with restriction enzyme

electrophoresis is run and DNA is denatured

DNA transferred to nitrocellulose paper and label is added

Probes hybridize to signal sequences

IDs fragments by size and sequence
-allow discrimination between alleles if mutation has changed the size of the DNA fragment

What does it stand for?
When is it used? (2)
Does mutation need to be known prior?

Restriction fragment length polymorphisms

used when mutation has caused a loss of restriction site

used to study families with mutations

Do not need to know the precise nature of the mutation

Deletion detection using southern blot

How do deletions appear in heterozygous individuals?
Explain this appearance.

deletions between 2 restriction sites
-larger deletions will cause fainter bands in heterozygous individuals b/c there is less target DNA for probe to anneal to

Hemophilia A and southern blot

What causes type A?
why is it able to be tested for using southern blot?
What is the most common mutation leading to type A hemophilia?
what kind of probe should be used?

Severe type caused by intrachromosomal rearrangement of F8 gene.
-gene is duplicated and inserted in the opposite direction causing a rearrangement in the length

F8 is the most common mutation leading to Hemophilia A
-should be checked for using RFLP

Northern blot

what does it measure?

Detects RNA species based on size and sequence complementary.

Also can measure abundance.

what does it stand for?

what is the end result?

How much DNA is required?

Do you need to have prior information about sequence?


Polymerase chain reaction

in vitro amplification of specific DNA sequences

Requires very little genomic DNA

Requires some sequence information

Sequential rounds of DNA replication at high temperatures followed by cooling.

Ingredients needed for PCR amplification (5)

Templet DNA




Thermostable DNA polymerase (taq)

Uses for PCR

Allows studying of small portions of the genome in isolation by amplification of primers

allows the analysis of effects due to single nucleotide change in detail (RFLP, mutation determination, sequencing)

can detect size variations in 1-1000bp range

IDing the HB-S (sickle cell) allele by PCR and restriction digest

HB-S mutations that alter restriction enzyme sites

If restriction sit is lost, fragments get longer.

Appears as multiple bands in the gel

Single base change in β globin gene

can design probes that match the normal and mutant, but must know the mutation

Use of oligonucleotide labeling (3)

what kinds of probes are preferred? why?

detecting single base changes when the restriction site is unchanged

used as primers in PCR

used as probes in ASO

**Conventional probes are preferred b/c they are usually more stable due to their length


what is it?
what does it descriminate against?
does mutation have to be known?

what diseases is it suitable for screening?

Allele specific oligonucleotide

usually single exon is amplified by PCR

discriminates between sequences differing by a single base
-requires that mutation must be known

suitable for screening:
CF, β-thalassaemia, ashkenazi mutations

PCR and turner syndrome

turner syndrome is caused by 45X

there is a risk of small amount of 46XY cells in gonads which can turn malignant

PCR for Y specific sequences will detect it

3 molecular diagnostic methods by PCR

1- restriction digestion of PCR amplified DNA and checks size on gel
--only when mutation abolishes or creates a restriction site

2. Hybridize PCR- amplified DNA to allele specific oligonucleotide on dot blot
--general method for specified point mutations
----large arrays allow scanning for any mutation

3. Check size of expanded repeat
-for dynamic repeat distance only (smaller ones--large expansions require southern blot)

what does it stand for?

what does it detect?

what probes are often used?

Good tool to identify what genetic mutations?

when will FISH not descriminate?

what diseases can FISH be used to detect?

Florescent In Situ Hybridization

used to detect large deletions

bacterial artificial chromosome (BAC) probes often used

If the probe is larger than the deletion, it will not descriminate

Good to identify multiple congenital anomalies, translocation, and trisomies

Used to detect
Williams syndrome, DiGeorge syndrome, Prader Willi, and Anglemann

FISH mechanism

Purified clone is made

allowed to anneal with chromosome on slide

exposed to UV

DeGeorge Syndrome

What causes it?

22a11 deletion syndrome

Short stature
Cleft palate, flat nose
slim hyperextensible limbs
mild mental retardation


how do you tell deletions vs duplications?

comparative genomic hybridization

Use to look at the entire gene

Plate with 2 colors

Ratio of green to red is plotted on a graph
-ratios above normal limit--duplication
-ratios below normal limit-- deletion

DNA sequencing

necessary when (3)

Gold standard for mutation ID

Necessary for Autosomal dominant conditions
ie: Marafan syndrome and collagenopathies

used in family specific mutations

and recessive conditions w/o common mutations
ie: atypical CF

Nail Patella syndrome

inheritance pattern?

how does it show up on gel?

what is the difference between R200Q and R198X?

Autosomal Dominant
--means mutation can be heterozygous

on gel shows up as 2 bands in the same position

2 kinds

ARG--> GLY causing loss of Mspl site

ARG-->Termination codon

Using sequence with β thalassaemia

what does the sequence ID?

what causes β Thalassaemia?

can ID β globin mutation in each individual

β thalassaemia is due to a null mutation in the β globin gene

-causing a stop codon


what causes it?

where is mutation generally located?

what test is used for identifing LOHN?

Caused by single base change in mitochondria DNA
--change is where restriction enzyme would cut
changes fragment length
----this makes PCR possible

stands for?

do you need to know mutation?

what does this test depend on?

single stranded conformational polymorphism

when the exact mutated gene is unknown

depends on the tendency of DNA to form hairpin loops
-looking at secondary structure

SSCP mechanism

used to ID mutation in what disease?

amplified by PCR
non-denaturing gel so renaturing does not occur w/ complementary strand

single stranded DNA migrates on gel

during migration, DNA anneals to itself-->hairpins

allows migration at different speeds

used to ID mutations in CFTR

advantages of SSCP?

Disadvantages of SSCP

-screen multiple exons from multiple samples at once
-moderate sensitivity

-not all point mutations affect secondary structures
-preferential ID of small insertions/deletions

almost obsolete technique

Common types of DNA polymorphism (3)

CA-repeat marker- measures # of CA repeats
--provides the most information

Single nucleotide polymorphism (SNP)- recognizes single base change
--most common

RFLP- specific SNP

CA repeat markers

what kind of primers?
seperate based on?

what can the be used to discriminate between?

radio labelled primer

primers are like ASO

separate purely based on size
-categorized based on how many repeat units are present

used to tell if chromosome came from mother of father

RFLP uses

how are they categorized?

categorized by if they are present or not

only 2 possibilities: there or not

not good to determine which chromosome came from which parent

Use what test?
big changes in sequence?
deletions of 100s-1000s kb?
single change in nucleotide?
between related genes?

big probe
southern blot

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