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CNV, NGS
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Copy number variation New genome sequencing Techniques to Identify Mendelian and Multifactorial Disorder Genes
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containing a reporter group or labeled group such as a biotinylated nucleotide or fluorophore; or aphage promoter to drive gene expression.
Mismatch primer mutagenesis:
Not preferable to do pcr in this way because high mistakes will appear classical in vetro
mutagenesis is preferable method in mismatched primer mutagenesis, the primer is designed to be
only partly complementary to the target site, but in such a way that it will still bind specifically to
the target, Inevitably this means that the mutation is introduced close to the extreme end of the
PCR product, This approach may be exploited to introduce an artificial diagnostic restriction
enzyme site that permits screening for a known mutation. Mutations can also be introduced at any
point within a chosen sequence by using mismatched primers. Two mutagenic reactions are
designed in which the two separate PCR products have partly overlapping sequences containing
the mutation. The denatured products are combined to generate a larger product with the mutation
in a more central location.
Develop 4 primers:
- Two inner primers containing a mismatch at the base(s) where you want to introduce the
mutation, in both directions
- Two regular outer primes upstream and downstream of the mutation
Perform two separate PCR reactions, both combinations with one regular and one mismatch
primer.
Combine both PCR products and remove primers.
Denature and renature: Some of the renaturations will be heteroduplexes of both PCR products.
Add DNA polymerase and dNTPs to complete heteroduplexes.
Dilute the reaction, and use as a template for a new PCR reaction with both outer primers he
resulting PCR product contains the mutation.
You need to sequence the PCR product to verify if no additional mutations have arisen during
PCR.
Chapter 4: CNV
There is some variation in the Human Genome, which makes us all different.
Single nucleotide polymorphisms
, For a long time, we thought that the major source of human variation could
be explained by SNPs (= single nucleotide polymorphisms). A SNP is
defined as a substitution of a single nucleotide that occurs at a specific
position in the genome. Those variations are common in the population: on
average there is 1 SNP/700 bp (discovered in the 1000 genome project).
Other types of genomic variation
CHROMOSOMAL DISEASES:
Down syndrome – Trisomy 21
Trisomy = an extra (3rd) copy of a chromosome present in the cell nuclei. This is structural
variation of the genome. Trisomy 21 = extra copy of chromosome 21.
Monosomy = only one copy of a chromosome in the cell nuclei.
Prader-Willi syndrome
This chromosomal disease is caused by a loss of function in genes in a particular region of
chromosome 15, caused by a deletion (very small region is actually missing).
Phenotype: overweight (uncontrollable eating pattern), mental retardation, small hands and feet.
Techniques for recognition variation when looking for chromosomal abnormalities:
FLUORESCENT IN SITU HYBRIDISATION (FISH)
Back then, when Prader-Willi syndrome was discovered, it was only found in a karyogram (you
couldn’t do much else that time). A better way of diagnosing such chromosomal diseases is with
Fluorescent in situ Hybridisation (FISH).
It’s a molecular cytogenic technique that uses fluorescent probes that bind to only those parts of a
nucleic acid sequence with a high degree of sequence complementarity.
How does it work?
You isolate a part of the chromosome that you’re interested in. You then denature it to
make it single stranded and label it with a fluorescent dye. Then you hybridize it back
to the position it recognizes in the chromosome. You will get a probe signal on your
chromosome.
With Prader-Willi, you won’t see a probe signal because that part of chromosome 15
won’t be there. Then how will you diagnose it? You just stain the entire chromosome
and see where no hybridization took place (look for spots where the probe signal is
missing you’ll know there’ll be a deletion there).
, Advantage: it’s a very reliable technique
Disadvantage: but you can’t do this with multiple, like 100s, of chromosomes at a time.
ARRAY-CGH
A solution to the disadvantage of FISH, is Array-CGH, where you can do multiple samples at a
time (at a wider scale). CGH = Comparative Genome Hybridization. It’s commonly used in
genetic diagnostics (since CNV’s are a frequent cause of genetic diseases). They’re designed to
screen a series of probes.
It’s a molecular cytogenic technique for the detection of chromosomal copy number variations
(CNV’s) on a genome wide scale.
How does it work?
You start with 2 genomic DNA populations
• Test sample (DNA you want to analyse)
• Control sample (reference DNA)
These samples are labelled with a different fluorescent dye (e.g. Test = green and
Control = red)
Then the samples get mixed
The mixed samples are then hybridized onto a panel that contains non-labelled DNA
probes which represent the genome
By comparing the patterns of hybridization of both samples, you can screen for changes
in copy number of large regions of the genome.
• So in the normal regions: the same ratio for both samples. If the test sample has
the same amount of copies in comparison to the reference/control sample, you’ll
see a yellow fluorescent signal (50% red and 50% green)
• In regions with CNV, the ratio between both samples will differ. If the test
sample has more than 2 copies = green. If the test sample has less than 2 copies
= more control sample = red.
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