Cycle 10 Workshop Flashcards
What are the components of PCR?
- Primers
- dNTPs
- Taq polymerase
- MgCl2
What does PCR stand for?
Polymerase Chain Reaction
List:
The functions of the components of PCR
- Primers: Extend new strand
- dNTPs: 4 nucleotides
- Taq polymerase: Adds dNTPs
- MgCl2: Mg as important pol. cofactor
What are the steps of PCR?
- Denature + separate strands (high temperature, ~ 95 degrees)
- Anneal primers (~55 degrees)
- Taq polymerase binds to DNA and starts synthesis (~72 degrees)
Define:
Multiplex PCR
Uses different primers for multiple sequences
True or False:
Each step in PCR is timed
True
What is PCR based on?
DNA replication
What does RT-PCR mean?
Reverse Transcription PCR
List:
Steps of RT-PCR
- Lyse cells: Release proteins, genetic material
- Extract RNA phase: Treat with DNase
- Target Poly-A tail: Oligo (dT) primer)
- Reverse transcription: Reverse transcriptase
- Degrade mRNA: RNase activity (reverse transcriptase)
- PCR - Synthesize DNA: Taq polymerase
What is the difference between cDNA and gDNA?
cDNA is produced by RT-PCR and contains no introns
What does cDNA stand for?
Complementary DNA
True or False:
cDNA only contains expressed genes
True, is does not have introns
True or False:
cDNA can exist in our cells
False, cDNA does not exist in our cells
What are the 2 main functions of RNase?
- DNA polymerase activity: Requires oligo-DT primer to start
- RNase activity: Degrades the mRNA from the RNA/DNA hybrids
For gDNA:
- What does it stand for?
- What does it contain?
- Where does it exist?
- How is it obtained?
- Genomic DNA
- Contains both introns and exons
- Exists in the nucleus
- Obtained using polymerase
For cDNA:
- What does it stand for?
- What does it contain?
- Where does it exist?
- How is it obtained?
- Complementary DNA
- Contains only exons
- Does not exist in human cells
- Obtained using reverse transcriptase
Define:
STRs
Short Tandem Repeats
* Highly polymorphic (unique)
* Short sequences of DNA with variable repetition
* CODIS (FBI database) uses 13 STRs loci in DNA profiling
True or False:
Children inherit number of STRs from one parent
False, children inherit one allele from each parent at a single STR locus
How are STRs analyzed?
With gel electrophoresis or capillary electrophoresis
What is compared in an STR electropherogram?
- Standards and samples
- Heterozygous vs. Homozygous
How is PCR for STR locus done?
- Primers that will anneal to the sides of the STR are created
- Primers will then replicate the section in the middle
- Collect the products and run electrophoresis
Define:
Electropherogram
A graph obtained by capillary electrophoresis
What is the difference between capillary and gel electrophoresis?
Capillary electrophoresis is the same as gel electrophoresis, just through a tiny tube
How is sex determination done with STRs?
Looking for AMEL to determine sex
* Shorter in X
* Longer in Y
What is one application of PCR?
Creating cDNA which is expressable in prokaryotes (bacteria) (e.x. for producing insulin)
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
Describe:
The CRISPR locus
Has spacers between short palindromic repeats - DNA sequences from previous phage infections (adaptive immune system of bacteria)
What is Cas 9?
Cas9 is a protein isolated from bacteria
Describe:
Phase 1 in CRISPR
- Cas1 and Cas2 cut viral DNA
- Store into CRISPR locus in bacterial genome (spacers between palindromic repeats
Describe:
Phase 2 of CRISPR
- Transcribe CRISPR locus into pre-crRNA
- Binds to tracrRNA (hybrid) (process with RNase III)
- Binds to Cas9 to create complex
- Signals CRISPR-Cas9 to find viral sequence and cut
What guides CRISPR to target gene that needs to be edited?
Guide RNA/CRISPR-Cas9 hybrid
What does Cas9 need beside sequence to be recognized by guide?
PAM (protospacer adjacent motif) - NGG - Sequence
Define:
NHEJ
Non-Homologous End Joining
* Disruption caused so that gene is not functional
* Error prone
Define:
HDR
Homology Directed Repair
* DNA strand looks for homology, can provide donor DNA with necessary edits (correction or add mutation/gene)
* Not efficient
List the 4 components of base editing of a single strand
- d/nCas: Dead nic, makes single-strand cut
- Guide RNA
- Cytidine deaminase: Attached to Cas9
- Uracil glycosylase Inhibitor (UGI)
How CG changed to TA?
- Cytosine demainase changes C to U
- UGI prevents the cell from changing U back to C
- Now have a UG, nic to remove G and add UA
- Through DNA replication or repair UA changes to TA
List
Applications of CRISPR inside and outside the body
Inside
* Ocular disease (in vivo)
Outside
* Cancer (designer T-cells)
* Blood disease
Why can’t normal T-Cells attack cancer cells?
Normal T cells contain PD-1 (protects tissue from autoimmune attack and facilitate tumor progression
