Genetics final

Question 1

PCR

Answer 1

amplifies a part of DNA

Question 2

RT- PCR

Answer 2

PCR with addition of a reverse transcriptase. amplifies cDNA that was transcribed from mRNA

Question 3

qRT-PCR

Answer 3

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.

Question 4

PCR vs RT-PCR-

Answer 4

RT-PCR has addition of reverse transcriptase. RT- PCR is on RNA and PCR is on DNA

Question 5

RNA- sequencing

Answer 5

a way to understand and determine gene expression patterns

Question 6

RNA- sequencing process

Answer 6

1. isolate the RNA you are interested in looking at
2. convert RNA to cDNA with reverse transcriptase
3. cDNA is broken into fragments
4. adapters are added to the end of fragments to prepare for sequencing and amplification
5. DNA is amplified with PCR
6. DNA is sequenced and then mapped.
7. 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.

Question 7

situ hybridization

Answer 7

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.

Question 8

Immunostaining (+ how is itt different from in situ hybridization)

Answer 8

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

Question 9

Explain how CRISPR-Cas provides immunity for bacterial cells

Answer 9

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

Question 10

Explain how homology searches can provide information about possible gene function

Answer 10

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

Question 11

Orthologs

Answer 11

genes evolved from same gene

Question 12

Paralogs

Answer 12

genes evolved by duplication

Question 13

posttranscriptional gene silencing (RNAi)

Answer 13

destabilize target mRNA and cause its rapid destruction, limiting the amount of protein that can be produced ⇒ known as “knockdown technique”

Question 14

siRNA

Answer 14

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

Question 15

miRNA

Answer 15

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

Question 16

Knockdown

Answer 16

Destabilizes target mRNA and causes its rapid destruction, limiting the amount of protein that can be produced. Uses RNAi and only reduces expression

Question 17

Knockout

Answer 17

Knockout involves the complete elimination or inactivation of a specific gene in an organism’s genome. Uses techniques like CRISPR-Cas9

Question 18

Transgenic animals

Answer 18

can be engineered to overexpress a particular gene, express a mutant form of a gene, or even lack specific genes.

Question 19

transgene expression

Answer 19

By observing the phenotypic consequences of transgene expression, researchers can infer the function of the gene and its role in biological processes.

Question 20

Targeted mutagenesis

Answer 20

involves introducing specific mutations at predetermined locations within an organism’s genome, often using techniques like CRISPR-Cas9 or gene targeting.

Question 21

Limitations of CRISPR Cas

Answer 21

1. Off-targeting
2. On-target but modification is not what we want (NHEJ instead of HDR)
3. PAM requirements; If the target DNA sequence doesn’t have the right PAM sequence, CRISPR-Cas9 won’t be able to make the desired edits in that area.
4. Delivery of the tools of gene editing to the tissue (sometimes it requires injection into adult germ-line).

Question 22

Advantages of CRISPRcas

Answer 22

1. Therapeutic applications; CRISPR-Cas gene editing holds promise for treating genetic disorders and diseases.
   * Efficiency; CRISPR-Cas systems are highly efficient at inducing DNA breaks and promoting DNA repair processes.
   * Precision: CRISPR-Cas systems can target specific DNA sequences with high accuracy.
   * Versatility: Allows for a wide range of applications in gene editing, such as correcting mutations and regulating gene expression / transcription.