Genetics final Flashcards
PCR
amplifies a part of DNA
RT- PCR
PCR with addition of a reverse transcriptase. amplifies cDNA that was transcribed from mRNA
qRT-PCR
a way to quantify RT-PCR data. It does this through fluorescence and as cDNA is copied, the fluorescence increases. As the fluorescence increases, it reaches the Ct value, which is the threshold. Thus, the lower the Ct value, the higher mRNA there is.
PCR vs RT-PCR-
RT-PCR has addition of reverse transcriptase. RT- PCR is on RNA and PCR is on DNA
RNA- sequencing
a way to understand and determine gene expression patterns
RNA- sequencing process
- isolate the RNA you are interested in looking at
- convert RNA to cDNA with reverse transcriptase
- cDNA is broken into fragments
- adapters are added to the end of fragments to prepare for sequencing and amplification
- DNA is amplified with PCR
- DNA is sequenced and then mapped.
- Since there is so much data present for this, RNA sequencing is often shown in heat maps. Heat maps can make it easier to see the patterns and correlations of data.
situ hybridization
In situ hybridization is a method to see RNA or DNA in tissues by inserting labeled DNA or RNA to complement a sequence. may be used to research and profile DNA or RNA sequences.
Immunostaining (+ how is itt different from in situ hybridization)
Immunostaining is marking antibodies to view proteins. They are different because they are targeting two different things (DNA/RNA vs proteins). may be used in things such as diagnosing or researching an illness
Explain how CRISPR-Cas provides immunity for bacterial cells
It retains a memory of the previous infections. Short sections of the infection are inserted into CRISPR with repeated sections inbetween. Then it is transcribed into RNA, which then is created into individual cDNAs and they search for matches of new viral DNA. if there is a match, it destroys and blocks it with Cas protein
Explain how homology searches can provide information about possible gene function
Homology searches are DNA and protein comparisons from different species and different species. This can help with determining the function from the sequence and the likelihood of sequences being related evolutionarily
Orthologs
genes evolved from same gene
Paralogs
genes evolved by duplication
posttranscriptional gene silencing (RNAi)
destabilize target mRNA and cause its rapid destruction, limiting the amount of protein that can be produced ⇒ known as “knockdown technique”
siRNA
Leads to degradation of mRNA
Original mRNA is chopped into smaller pieces called siRNAs
One strand of the siRNA enters a protein complex called RISC
siRNA acts as a guide for the RISC and when the siRNA binds to a complementary sequence on an mRNA, an enzyme in the RISC cuts the mRNA into two pieces which degrade in cytoplasm
miRNA
Leads to inhibition of translation
miRNA encoded from introns and exons of mRNA
Chopped into smaller RNA molecules
one strand of the miRNA enters a protein complex called RISC ⇒ inhibits translation