Genetics Unit 17: Clinical Applications Of Molecular Bio

Front 1

What is FISH used to detect?

Back 1

Aneuploidies and large chromosomal rearrangements or deletions.

Front 2

Advantage of FISH over standard chromosome staining

Back 2

Dont need to wait for metaphase, can do during interphase. Also allows rapid determination of the identity of rearranged chromosome material or fragment.

Front 3

Limitations of FISH

Back 3

Mutations of just a few nucleotides wont be detected.

Front 4

What can genomic screening (micro array) be used for?

Back 4

1) Determine carrier status for lots of human diseases and predispositions to cancers.
2) Can learn what specific strain of HIV is infecting a patient or which oncogene/tumor suppressor genes are defective in cancer for more precise treatment.

Front 5

FISH process

Back 5

1) Start with chromosomes on a glass slide (metaphase or interphase).
2) DNA is denatured (heat or alkaline).
3) Probe is made, flourecently labeled, and denatured.
4) Probe added to chromosomes
5) Probes find their complimentary strand and form a hybrid.
6) Florescence tells whats there.

Front 6

Micro array process

Back 6

1) Normal cell and cancer cell DNA (for example) are amplified by getting mRNA and then creating cDNA
2) cDNA for different cells is stained with different colors.
3) DNAs are put into wells which have specific complimentary DNA sequences
4) Analyze, if a pair of wells has both colors, no dif in good or bad cell. If only one color shows up, either gene is turned off or over expressed in bad cell.

Front 7

Pros and Cons of PCR

Back 7

Pros: Can use minute quantities of DNA and obtain large quantities within hours which can be cloned or analyzed. Is also very specific.
Cons: Only relatively short sequences can be amplified and one must know what the DNA sequence in question is.

Front 8

Multiplex PCR: what is it and give example of when its used.

Back 8

Using several PCR primers in the same reaction.
Used for diagnosing muscular distrophy. Is an X linked disorder in the largest gene in the genome. Therefore there are lots of exons (79) which could be faulty. Use multiplex PCR to scan all exons.

Front 9

Process of PCR

Back 9

Small amount of DNA is is added to a solution along with specific primers and DNA polymerases. First, the DNA is denatured with temp, second primers attach to sites, third, DNA pol. synthesizes a strand of DNA. Then renatured. This processes is constantly repeated.

Front 10

How does PCR detect expansion/reduction in a non coding region of DNA? Give an example

Back 10

2 primers are set to the left and right of the repeated region in question. When the PCR runs, will create products of a certain length. Length can be tested via gels. If there is a product that is much larger than a normal person, u know the region has undergone expansion.
Ex. EPM1

Front 11

SNP

Back 11

Single neucleotide polymorphisms. To be considered a SNP, must be present in at least 1% of the population.

Front 12

How is PCR used to locate single base pair alterations in DNA?

Back 12

Use primers whose 3' end terminates with the nucleotide in question. Therefore if one makes a primer that needs a normal final 3' basepair to work, an abnormal primer would have an incorrect binding on the last nucleotide and would not lead to DNA synthesis.

Front 13

RNA interference definition and mech.

Back 13

RNA interference is the way which eukaryotic cells sense double stranded RNA, chop it up, and attack any mRNA with the same sequence as the small bits its chopped.
1) Double stranded RNA is sensed and chopped up by an enzyme called Dicer
2) A complex called RISC then binds these small RNA bits (siRNA) and discards one strand (making them single stranded.
3) This activates the RISC complex by giving it its template. It then goes to mRNA with a complimentary template to its siRNAs and inactivates or destroys these mRNAs.
Has great potential in fighting viruses.