Genetic Conditions & Testing Flashcards
Describe PCR (Draw)
Polymerase Chain Reaction is a way to go from not much DNA to a ton of DNA, which is really valuable for research and genetic diagnostic tests. You use Taq Polymerase, a polymerase that can handle higher temperatures where DNA is denatured (split into single strands). Small primers, single stranded small bits of DNA, are laid down (you usually have to know what these are for it to work) and Taq polymerase adds more nucleotides to each side. This happens again and again until you have buckets of DNA.
What are the major methods of DNA sequencing/testing?
Sanger Sequencing (use PCR with dideoxyribose nucleotides used every once in a while. These stop replication and allow you to sequence every nucleotide in the sequence) Used to detect single base changes as well as insertion or deletion of small chunks of DNA
Genome-scale sequencing (or exome scale sequencing, it’s more efficient by only looking at exons) is good for looking at broad categories like hearing loss or developmental disabilities.
Southern Blot (look for specific strand of DNA)
Karyotyping (requires viable cells, allows you to visually look at entire genome)
Fluorescence in situ hybridization (FISH) looks at 100kb or smaller sequences, requires a specific target.
Microarray: look at entire genome for lost or gained alleles
Describe how molecular genetic testing is relevant for Cystic Fibrosis and Hereditary Breast/Ovarian cancer
Based on a person’s ethnic background you have a good idea of whether to test them for cystic fibrosis genetic errors or not. This can help them decide on having kids. With breast cancer, finding a breast/ovarian cancer gene informs the rest of the family and allows you to potentially identify cancers well before they would be noticed in routine checkups.
What are some issues with with interpreting microarray DNA sequencing results?
You don’t see position of errors, just what the error or difference is. So if there’s a balanced translocation you wouldn’t be able to notice.
What are some limitations of interphase FISH?
You need to know exactly what you’re looking for, and it doesn’t tell you anything about position, so translocation isn’t going to be found this way. And FISH only works on smaller genetic analyses, not on the whole genome.
What are some limitations of metaphase FISH?
You need viable cells, and you need to know what you’re looking for. But it tells you location and presence of sequence! FISH has a size limit of 100 kb
When could you use interphase FISH?
If you only have nonviable cells to look at, and you want to see if there’s an absence or extra sequence (you know what sequence to look at)
When could you use metaphase FISH?
You have viable cells, you want to know if there’s an absence or extra sequence (you know what to look at) and you want to see where the absent or extra sequence occurs
When could you use microarray DNA sequencing
When you want to test the whole genome for specific, small errors (missing genes or pieces of genes), or you have no idea what’s going on. You can search thousands of genes at the same time. Or look at genetic expression as a response to pathogens (general)
When could you use karyotyping?
When you’re looking for structural changes in chromosomes, missing chromosomes, translocated chromosomes, extra chromosomes
What are some drawbacks of karyotyping?
Expensive, doesn’t tell you what’s missing, but is more of a qualitative, high-level view of what appears to be different in a genome
What are limitations for PCR?
You can only efficiently do this for sequences less than 1000bp in length, and you need to know exactly what DNA sequence you’re looking to multiply.
Describe targeted analysis vs. gene sequencing.
Targeted analysis: you know the mutations or genes of interest. There are fewer aspects you need to consider. It’s cheaper and faster because you’ve narrowed your scope.
Gene sequencing: when there’s a problem but the cause is unknown. Mutations occur throughout the gene, or even the genome! More expensive, slower, and interpretation is tricky because there are millions of benign “mutations” or differences from a reference genome.
Other terms for large scale genomic analysis
Shotgun sequencing, genome sequencing, massive parallel sequencing, next generation sequencing
What are some difficulties with whole genome analysis?
The reference isn’t a golden standard, so there are millions of differences that don’t matter. Any given person will have 3 million variants. 100,000 variants will occur within genes. 25,000 occur in exons or near exons. 1000-5000 associated with diseases, 99% of these are still benign mutations.
What does VUS stand for?
Variants of uncertain significance (we don’t know if these mutations matter, or how they change expression) “it is common to find a rare variant”
What is the 5-class scale for pathogenicity?
1 (benign) 2 (likely benign) 3 (variant of uncertain significance) 4 (likely pathogenic) 5 (pathogenic) 4 and 5 are deemed “positive” results
What is genetic linkage?
Genetic linkage is a modern argument against Mendel’s law of Independent Assortment (this is the idea that each allele is independent of other alleles). Linkage is the idea that genes close to each other on a chromosome are more likely to be expressed in a pattern.
What is linkage disequilibrium?
Non-random association of alleles within a population. If two alleles present more commonly together than expected from independence, you get linkage disequilibrium
What is linkage disequilibrium?
Non-random association of alleles within a population. If two alleles present more commonly together than expected from independence, you get linkage disequilibrium
What would a fully linked system look like?
Dominant would go with dominant, recessive would go with recessive, no combinations
What is electrophoresis?
Apply a charge gradient to a gel and watch substances (if controlled for charge/surface area) will move through the gel at speeds dependent on size. DNA is uniform regardless of size, proteins will be denatured with SDS PAGE to guarantee uniform charge-to-mass ratio
How do you read a blot? Draw
Same for western, northern, southern, just different molecule type being analyzed. Look for presence or absence of bands at different weights based on what you know about the molecules.
Describe the types of chromosome abnormalities that are routinely detected using both traditional and molecular cytogenetic techniques.
you can find translocation (balanced with karyotyping or metaphase FISH, unbalanced with karyotyping, metaphase FISH, interphase FISH, and microarrays) you can find deletions and duplications of genes (FISH, microarrays) or whole chromosomes (karyotyping or microarrays)
Describe some of the phenotypic and reproductive risks associated with chromosome abnormalities
If you have a chromosomal abnormality there is a chance you’ll pass it on to your kid, since they’re getting their chromosomes from you. Considering phenotypes, over-expression of genes from duplicates causes a lot of trouble, and missing genes from deletion can be even worse. Monosomy is when you’re missing a chromosome. Trisomy is when you have one extra
What are the two types of chromosomal abnormalities? What is a condition caused by each type?
Structure and number abnormalities. Down Syndrome is a number abnormality. Philadelphia chromosome seen in some leukemias is a structure abnormality
What is the p arm of a chromosome? the q arm?
P arm is “petite” smaller arm, Q arm is bigger
What are some limitations to karyotyping?
Expensive, limited by resolution greater than 3-5Mb. Sometimes hard to know exactly what’s missing (crossover of genes and such)
What is gene product alteration/fusion?
If the regulatory region of one gene is fused with the functional region of another gene you may get an unexpected amount of expression from a gene (example, not rule)
What phenotypes (physical manifestations) are associated with chromosomal abnormalities?
Cancer, Abnormal growth or development in children (developmental disabilities, birth defects), early death (spontaneous abortion often indicates translocation or monosomy)
What is one way a carrier of a genetic condition passes along that condition to their offspring?
A carrier may have a balanced translocation error. So they have all of the DNA they need. But their offspring will receive only one of the chromosomes, so they’re almost guaranteed to have some trouble.
