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Flashcards in Molecular genetic diagnostics Deck (11):

What is FISH, or Flourescent in situ hybridisation?

It is a cytogenic technique (focuses on the structure and function of the cell), that uses fluorescence to reveal multiple copies of the same chromosome that may be present in the organism. It is used on fetal cells to search for chromosomal abnormalities. It is used to search for the presence of down syndrome. If the fetus does have down's then the three copies of the chromosome 21 will glow with identical colours.

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Can people still have translocated chromosomes, or rearranged DNA without any physical abnormalities?

Yes, they can-this is called reciprocal translocation. If one half of a chromosome jumped off and joined another chromosome, provided that in the entire set of chromosomes there was still only 1 copy of that chromosome, then that individual would be fine. The rule of thumb is that as long as there is no net gain or loss of genetic material, they should be fine.

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What is robertsonian translocation?

This form of translocation is rare, but it does cause problems in the individual.

Robertsonian translocations can only occur between acrocentric chromosomes. Acrocentric chromosomes are the ones that have one long arm and the other arm is so short it barely looks like it's there. These are chromosomes 13,14,15,21 &22. 

They hop ontop of each other instead of staying seperate and cause a robertsonian translocation. Provided that the translocation is reciprocal, then it is fine. 



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What is the difference between a balanced versus unbalanced translocation?

The balanced translocation is reciprocal, and there is no net gain or loss of genetic material in the child. 

However, an unbalanced translocation is when a one parent has given their child a robertsonian translocated chromosome with 2 chromosomes on one, and the other parent has given the child a normal set of chromosomes. That means the child could will end up with 3 copies of one chromosome, which is unbalanced and in the case of down's syndrome, will cause trisomy 21. 

Unbalanced translocations can be gains or losses of chromosomes. 

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What are DNA microarrays and how do they work?

The DNA microassay is a way of chopping up DNA strands into microparticles that fit into the microscopic slips in the microarray disc. Then the microarray chip assays the individual DNA particles bit by bit. This microarray means that more genes can be identified and that the entire chromosome can be examined much faster. 

They can pick up so much detail that they can detect single nucleotide polymorphisms and copy number variations. 


What can chromosome microarrays detect in a DNA sequence?

They can detect single nucleotide polymorphisms, such as deletions, insertions and missense mutations in which the 4 base pairs in the DNA sequence have errors in them. 

Deletions in particular are very easy to detect in microarrays. There is a characteristic dip in the produced graph, that indicates the absence of a particular basepair or base itself. 

In the case of wolf-hirschhorn syndrome, there is a deletion at the 4p terminal end that is picked up on the microarray graph. 

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What are the main techniques used to detect single gene disorders?

Polymerase chain reaction (PCR) are when they blow up and amplify specific DNA sequences of interest around the site of mutation so that they can confirm the presence of a mutation.

Restriction fragment polymorphisms use restriction enzymes that cut specific sequences of DNA. The restriction enzymes can only cut at certain base pairs, and so the presence of a mutation would not allow the enzyme to make a cut there and thus the mutation would be identified/confirmed.Increasingly DNA microarrays are also being used to detect single gene mutations too. 


How are PCR and restriction enzymes techniques both used to detect the beta-globin mutation?

First, the suspected DNA sequence is amplified using PCR. The nature of the beta-globin mutation involves a thymine being changed to a cytosine. This results in a loss of recognition of the NcoI restriction enzyme, that detects thymine bases. 

So, if the NcoI restriction enzyme is able to detect and cut the transcription start site, then the thymine has been recognised and the mutation is not there. If the NcoI does not recognise the site, then the mutation from thymine to cytosine has occured and the individual is positive for Beta-thelassemia. 


Under which circumstances or conditions is genetic testing conducted?

Clinical diagnosis when symptoms occur, carrier testing for couples wanting to start a family and who don't want to spread a hereditary condition. Then there is  presymptomatic/predictive testing for individuals who want to check forthe presence of any hereditary conditions. 


What is prenatal diagnosis?

When a couple expecting a baby do genetic testing on during the early period of the pregnancy. This gives them the choice to terminate the pregnancy, or make arrangements to support the baby and naturally the relief if their child is normal. 


What are the techniques used for prenatal diagnosis?

Chrionic villus sampling (when they inject a syringe into the womb and take placental cells for genetic testing). 

There is also amniocentesis (when they inject a syringe into the womb and take a sample of the amniotic fluid, because there are often sloughed fetal cells in it. These fetal cells are tested). 

Then there is pre-implantation genetic diagnosis, which requires IVF. After the fertilisation in the lab, a few of the zygotes cells are taken and tested before being injected back into the mother. 

Lastly, there is the cell free fetal DNA/RNA in maternal blood that can be taken via a blood test, filtered for the fetal cells and genetically tested.