mutations and genetic analysis

Question 1

Describe the types, effects and nomenclature of mutations

Answer 1

Types:
-numerical- ANEUPLOIDY
Trisomy 13-Patau syndrome-facial disfiguration and mental retardation. Non-dysjuction. maternal. Few survive first year.
trisomy 18- Edwards syndrome. Non-dysjunction maternal. Most die in first month.
Trisomy 21- Down syndrome- facial disfiguration, reduced life expectancy (50-60years), IQ less than 50, Alzheimers in old age. In most cases - non-dysjuction origin.
45,X- Turner syndrome.Due to absence of 1 X chromosome. In females-short and infertile, webbed necks same learning skills
47, XXY- males tall, infertile, gynaecomastia
-structural
-genetic:
missense: different amino acid
non-sense: stop codons
silent
frameshift: insertion/deletion
point mutations: trasversions-purine to pyrimidine
transitions-purine-purine

Question 2

PCR

Answer 2

PCR:

• DNA
• DNA polymerase
• Free nucleotides
• Oligonucleotide primers
• sequence information.
  method: heat to 95, anneal at 50-75, extend at 70-75. Repeat 20-30 times

Gel electrophoresis:

• separate DNA fragments
• apply electric field
• negatively charged DNA will move and separate out fragments.

Question 3

ARMS

Answer 3

Amplification Refractory mutation system. 
Wild-type gene
In normal primer- amplification 
In mutant primer- no-amplification
In mutant gene:
-Normal primer: no amplification 
-Mutant primer: amplification
Gel electrophoresis. 
Advantages: 
-cheap
-radiolabelling not required
 Dis:
-Need to know mutation you are looking for to design primer 
-limited amplification 
-untrustworthy replication of DNA

Question 4

RFLP

Answer 4

Restriction Fragment length polymorphism
-Uses enzymes from bacterial cells which cut DNA at specific sequences
-In gel electrophoresis will show different bands
Adv:
-uses gel electrophoresis
-does not require labelling
- simple/cheap

Question 5

DNA sequencing

Answer 5

Requires:

• modified nucleotides ddNTP
• normal nucleotides dNTP
• Dna polymerase
• Oligonucleotide primers
• pre-made solutions
• Dna strand

Copy strand of DNA using radioactively labelled modified nucleotides which will end the sequence at a specific base. 
Carry out gel electrophoresis.
ADV:
-gold standard
-Automated
-very good reading of first 40 bases 
Dis:
-very expensive 
-deterioration in reading of bases after 700-900 bases.