Mutations and Genetic Analysis Flashcards
Incidence of chromosomal abnormalities
First-trimester miscarriages = 50%
Children with mental retardation = 35-40%
Congenital malformations = 5-10%
trisomy accounts for
50% first trimester miscarriages
main trisomy include
other trisomy
trisomy 16
trisomy 21
monosomy accounts for
20% first trimester miscarriages
trisomy 21 is
downs syndrome
Monosomy 45 X means
only 45 chromosomes, one X being missing (this is Turner syndrome).
in liveborn infants most common is the abnormality
trisomy 21
45 X
Trisomy 47,XX +13 causes
Patau
Trisomy 47,XY +18 causes
Edwards
Trisomy 47,XX +21
downs syndrome
47,XXY causes
Klinefelter
in trisomy 13, 18, 21 and and 47XXY, origin of disjunction is usually from
maternal side
in monosomy 45X, origin of disjunction is usually from
paternal side
Unbalanced Robertsonian translocation (4%) is linked to
downs syndrome
Autosomal aneuploidy syndromes:
downs syndrome
Incidence: 1 in 650 to 1 in 700 Characteristic facial dysmorphologies IQ less than 50 Average life expectancy (50-60 years) Alzheimer’s disease in later life
Autosomal aneuploidy syndromes: Trisomy 13 (Patau syndrome)
Incidence: 1 in 5000
Multiple dysmorphic features and mental retardation
About 5% die within first month, very few survive beyond first year
Unbalanced Robertsonian translocation (10%) is linked to
Trisomy 13 (Patau syndrome)
Autosomal aneuploidy syndromes: Trisomy 18 (Edwards syndrome)
Incidence: 1 in 3000
Severe developmental problems; most patients die within first year, many within first month
Sex chromosomes aneuploidy syndromes:
45,X (Turner syndrome)
Incidence: 1 in 5000 to 1 in 10000 (liveborn)
Females of short stature and infertile
Neck webbing and widely spaced nipples
Intelligence and lifespan is normal
Sex chromosomes aneuploidy syndromes:
47,XXY (Klinefelter syndrome)
Incidence: 1 in 1000
Tall stature, long limbs
Male but infertile, small testes, about 50% gynaecomastia
Mild learning difficulties
Structural abnormalities
Balanced or unbalanced rearrangements Translocations Deletions Insertions Inversions
Reciprocal Translocations:
involving breaks in two chromosomes with formation of two new derivative chromosomes
Robertsonian Translocations:
fusion of two acrocentric (near end of one) chromosomes
no genetic information is lost: other translocations occur but do not lead to a viable fetus.
deletion mutation is when
breaks in a chromosome leads to part of genetic material being deleted.
- unbalanced rearrangement
inversion mutation types
pericentric
- breaks in chromosome involving centromere gets inverted
paracentric
- break in chromosome where part of chromosome gets inverted
both are balanced rearrangements
genetic mutations can be
Germline or somatic Gene disruption /disease-associated Polymorphism - No phenotypic effect - Frequency >1%
Types of mutations
Non-coding
Coding
Coding mutations
Silent – synonymous e.g. CGA (Arg) to CGC (Arg)
Missense e.g. CGA (Arg) to GGA (Gly)
Nonsense e.g. CGA (Arg) to TGA (Stop)
Frameshift – deletion / insertion e.g. CGA (Arg) to CCGA (Pro, then out-of-frame
mutations detection methods
Polymerase chain reaction (PCR) - In vitro technique
Gel electrophoresis
Restriction fragment length polymorphism (RFLP) analysis
Amplification refractory mutation system (ARMS)
DNA sequencing
What do we need for PCR?
Sequence information Oligonucleotide primers DNA Nucleotides DNA polymerase
in PCR denaturation occurs at
93-95 degrees C
in PCR annealing occurs at
50-70 degrees C
in PCR extend occurs at
70-75 degrees C
Gel electrophoresis
Separate DNA fragments by size Apply an electric field - direction of migration is from negative to positive DNA is negatively charged Separate through agarose gel matrix Visualise DNA fragments
Advantages of Gel electrophoresis
Speed
Ease of use
Sensitive
Robust
PCR applications
DNA cloning DNA sequencing In vitro mutagenesis Gene identification Gene expression studies Forensic medicine Typing genetic markers Detection of mutations
ARMS (Amplification Refractory Mutation System) and primers
normal primer causes amplification
mutant primer causes no amplification
ARMS (Amplification Refractory Mutation System)
Advantages
Cheap
Labelling not required
Electrophoresis required
Primer design critical
ARMS (Amplification Refractory Mutation System)
Disadvantages
Need sequence information
Limited amplification size
Restriction endonucleases are
Enzymes from bacterial cells
- Protective mechanism
- Degrade DNA of invading viruses
- Recognise specific DNA sequences
- Usually 4-8 bp
- Always cut DNA at the same site
RFLP analysis is
PCR and digest
RFLP Advantages
Simple
Cheap
Non-radioactive
RFLP Disadvantages
Requires gel electrophoresis
Not always feasible
DNA sequencing:
Sanger
Chain termination method
ddNTP has
h groups
dNTP has
OH group
DNA sequencing:
Advantages
Gold standard for mutation detection
Automation and high throughput
Next generation sequencing
- 18 billion bp in 4 days (about 6 human genomes)
DNA sequencing:
Limitations
Expensive equipment
Poor quality sequence read
- First part of sequence (15 to 40 bases)
- Deterioration after 700-900 bases
Choice of method
Direct test Quick and easy Cheap Sensitivity Specificity
