Lecture 22 Flashcards
what are Single Nucleotide Polymorphisms (SNP’s) and what are the 3 types of variants that can occur in them?
the small differences in a single nucleotide in an organism that are responsible for all genetic variation within a species
neutral, pathogenic, or predisposing SNP’s can occur
What are restriction endonucleases and what do they do?
enzymes isolated from bacteria that cut DNA at specific sites
How is agarose gel electrophoresis different from the electrophoresis that is used in SDS-PAGE?
the DNA is already charged in Agarose gel electrophoresis (it is neutralized prior to electrophoresis in SDS-PAGE)
What does DNA ligase do? how can you make this process occur more easily?
it “glues” DNA fragments together
if the ends being joined are compatible sequences, this process does not require DNA polymerase to fill in the “Staggered edge” and the process is much more easily completed
what is a key organism used when cloning genes? what role does it play?
bacteria
the DNA fragement that is to be cloned is added (via restriction nuclease and ligase) to the bacterial genome and the bacteria’s quick replication clones the fragment along with the rest of it’s DNA
list and compare the 2 types of DNA libraries. be sure to include the enzymes that each type uses.
Genomic libraries: includes exons AND introns
uses restriction nuclease
cDNA libraries: includes only introns (coding regions)
uses reverse transcriptase to produce cDNA copies of mRNA’s
What is PCR used for? what do you need to know before you use this technique?
to amplifiy isolated DNA regions
the sequence of the gene (otherwise you cannot ID which DNA fragment you want, out of all of the other fragments)
What 5 things do you need in order to conduct a PCR?
a Double stranded DNA sample
Primers (oligos)
Heat-stable DNA polymerase
Deoxyribonucleotides (dNTPS)
a Thermocycler
describe the step by step process of PCR
put dsDNA sample under high temp. to denature/separate the dsDNA strands
add your handpicked primers that flank the 2 ends of the DNA fragment you wish to replicate
once the primers anneal to the DNA, add all 4 types of deoxy Nucleotide Triphosphates (dNTPS)
Once the dNTPS’s are added, the Taq Poleymerase can synthesize copies of the DNA
Why is a thermocycler useful for PCR and how do you know how many DNA copies each round of PCR will yield?
thermocyclers can be set to heat and cool over and over for however many cycles are needed
even if you start with just one copy of the DNA, the more cycles you conduct, the more Copies are made (almost exponentially higher as you perform more and more cycles)
What is the advantage and disadvantages of PCR?
advantage: you can make a lot of DNA copies using a very small initial sample
Disadvantage: you must know the sequence of the gene, the process is error-prone, and if the sample is a contaminated DNA sample then the copies will also be contaminated (they are clones duh)
State an example of PCR being used to diagnose a disease. how does this work?
PCR can help diagnose HIV by amplifying the HIV particle in a sample of an infected patient’s blood to the point where it can be visualized via gel electrophoresis
What is quantitative PCR? how does it work?
PCR that uses a “probe” (usually a flourescent tag) to identify the copy number of a specific gene in 2 or more samples at a time
it uses a probe, in addition to the normal PCR process, that fluoresces each time the PCR cycle is complete
what makes quantitative PCR useful?
it can detect levels of an infective agent
it can determine level of gene expression (can be used to ID cancer activity in a sample)
what is the probe that is usually used for quantitative PCR?
a complementary oligo with a fluorescent tag.
