Transcript PCR and diagnostics II

TYPES OF MUTATION CAUSING HUMAN GENETIC DISEASE
Nucleotide substitutions (point mutations)
Missense mutations
Nonsense mutations
Spice site mutations
Frame shift mutations
Small, a few base pairs
Rearrangements
Deletions
Insertions
Duplications
Unstable repeat sequences
Large, can be many
kilobases
MUTATION
Mutations in
Coding regions
Mutations in regulatory
domains
Protein abnormal
Expression abnormal
Loss of function - common
Gain of function - rare
GENETIC TESTING IN HEMOPHILIA A
X-linked recessive, 1/10000 males
variable in severity
severe cases, spontaneous and life threatening bleeding
repeated episodes can cause joint deformity and crippling
treatable
Gene is large, many mutations, can look for specific common inversion
with a PCR based test.
• e.g. Alpha anti trypsin deficiency
•Disease leads to increased probability of
developing pulmonary emphysema
•Results from single base pair change at a known
nucleotide position
• Synthetic oligonucleotide probe that contains the
wild type sequence in the relevant region of the
gene can be used in a Southern blot analysis to
determine whether the DNA contains wild type or
mutant sequence
• Using the appropriate temperature the
complimentary sequence will hybridize but a
sequence with even a single mismatched base
will not.
E.g. Oligoligation assay
• Normal sequence pair at site AT, Mutant is GC
• 2 short oligonucleotides that are complimentary to one of the 2 native DNA strands
are synthesised
• Probe X has as its last base at the 3’ end the nucleotide that is complimentary to
the normal sequence. It does not hybridize well to the mutant sequence as there
is a mismatch
• Run test sample next to normal control
• Oligos hybridize
• When ligase is added the oligos bound to the mutant can’t ligate as have wobbley
base that is misaligned
• In order to see whether the single base mutation is present need to be able to
distinguish between ligated and non ligated (containing mutation)
• Probe X has a biotin residue or fluorescent molecule at the 5’ end, Probe Y has a
dioxygenin residue at the 3’ end (called PEO in diagram)
• After hybridization and ligation, DNA is denatured to release hybridization probes
and mix is transferred to wells coated with streptavidin. Biotin binds
• Unbound material washed away
• Anti dioxygenin antibodies coupled to alkaline phosphatase added to the well
• Substrate for AP added
• If oligos ligated get colour in well
• If oligos not ligated no colour
• Some genetic diseases can be attributed to multiple
mutations (together or separately) at multiple different
sites. PCR has played a tremendous role in making
diagnosis possible in a timely fashion.
E.g. BRCA 1
•Plays a role in hereditary breast cancer
• Very long gene
• Analysis is PCR based
• Don’t know specifically what you are looking for e.g. can
be any of many mutations in BRCA 1, not all yet
identified
• BRCA 1 has 24 exons that span a huge number of bases
• Most mutations have been found in Exon 11
BRCA 1 Exon 11
• 3500bases long
• Too long for convenient PCR, split into 3 pieces for
analysis
• Oligonucleotides incorporate a promoter so amplified
products end up with a promoter on the front
• 3 pieces are amplified and each used separately in a
transcription translation system
• Protein products are produced
• Run on PAGE to see if are the correct size (next to
normal controls)
• If incorrect size know there was a mutation
• Screening of the protein product allows screening of a
very large pieceof DNA when you don’t know specifically
what you are looking for
Exons 2-10
• ReverseTranscriptase PCR is used
• 2-10 includes many introns and far too long to amplify. If
start with RNA there are nointrons
• Use RT to get DNA, amplify with special oligo with
promoter sequences
• Use transcription translation system as before, look at
protein product size
Exons 12-24
• As exons 2-12
Exon 2
• Mutation found that is common in Ashkenazi Jews
• If know source sometimes just screen directly from
genomic DNA, and look at size on gel to see if different
from normal control.
Fragile X Syndrome
1/1200 males, 1/2500 females - most common form of X-linked mental
retardation
Varying degrees of developmental delay
Hypermobile joints
High arched palate
Long face
Large protruding ears
•Known to be caused by expansion of a CGG repeat in the 5’ end of the FMR-1
gene.
•The repeat is polymorphic in the general population 6-45 CGG repeats
•Carriers, male and female, unaffected carry 50 to 200 CGGrepeats
•Affected individuals carry greater than 200 CGG repeats
•When the repeat size is great than 200, expression of the FMR-1 gene is turned
off
•The presence of more than 200 CGG repeats in the FMR-1 gene contributes to
the
•fragility of the X chromosome and this can be observed cytogenetically.
FMR-1 CGG Repeat Southern blot
Affected
>200 repeats
Premutation
60-200
repeats
EcoRI
BssHII
*
EcoRI
CGGn