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
In situ hybridization
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
DNA vs oligonucleotides
-what sizes are used for each?
DNA- usually 2x stranded for sizes of 1kb-100s of kbs
Oligonucleotides- usually 1x stranded
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
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)
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
What causes it?
22a11 deletion syndrome
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
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
how does it show up on gel?
what is the difference between R200Q and R198X?
--means mutation can be heterozygous
on gel shows up as 2 bands in the same position
ARG--> GLY causing loss of Mspl site
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
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
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
-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
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
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