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My Packs: UWO (BIO 1002) > Cycle 10 Workshop > Flashcards

Cycle 10 Workshop Flashcards

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Biology 101 flashcards
1
Q

What are the components of PCR?

A
  • Primers
  • dNTPs
  • Taq polymerase
  • MgCl2
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2
Q

What does PCR stand for?

A

Polymerase Chain Reaction

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3
Q

List:

The functions of the components of PCR

A
  • Primers: Extend new strand
  • dNTPs: 4 nucleotides
  • Taq polymerase: Adds dNTPs
  • MgCl2: Mg as important pol. cofactor
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4
Q

What are the steps of PCR?

A
  1. Denature + separate strands (high temperature, ~ 95 degrees)
  2. Anneal primers (~55 degrees)
  3. Taq polymerase binds to DNA and starts synthesis (~72 degrees)
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5
Q

Define:

Multiplex PCR

A

Uses different primers for multiple sequences

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6
Q

True or False:

Each step in PCR is timed

A

True

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7
Q

What is PCR based on?

A

DNA replication

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8
Q

What does RT-PCR mean?

A

Reverse Transcription PCR

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9
Q

List:

Steps of RT-PCR

A
  1. Lyse cells: Release proteins, genetic material
  2. Extract RNA phase: Treat with DNase
  3. Target Poly-A tail: Oligo (dT) primer)
  4. Reverse transcription: Reverse transcriptase
  5. Degrade mRNA: RNase activity (reverse transcriptase)
  6. PCR - Synthesize DNA: Taq polymerase
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10
Q

What is the difference between cDNA and gDNA?

A

cDNA is produced by RT-PCR and contains no introns

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11
Q

What does cDNA stand for?

A

Complementary DNA

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12
Q

True or False:

cDNA only contains expressed genes

A

True, is does not have introns

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13
Q

True or False:

cDNA can exist in our cells

A

False, cDNA does not exist in our cells

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14
Q

What are the 2 main functions of RNase?

A
  1. DNA polymerase activity: Requires oligo-DT primer to start
  2. RNase activity: Degrades the mRNA from the RNA/DNA hybrids
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15
Q

For gDNA:

  1. What does it stand for?
  2. What does it contain?
  3. Where does it exist?
  4. How is it obtained?
A
  1. Genomic DNA
  2. Contains both introns and exons
  3. Exists in the nucleus
  4. Obtained using polymerase
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16
Q

For cDNA:

  1. What does it stand for?
  2. What does it contain?
  3. Where does it exist?
  4. How is it obtained?
A
  1. Complementary DNA
  2. Contains only exons
  3. Does not exist in human cells
  4. Obtained using reverse transcriptase
17
Q

Define:

STRs

A

Short Tandem Repeats
* Highly polymorphic (unique)
* Short sequences of DNA with variable repetition
* CODIS (FBI database) uses 13 STRs loci in DNA profiling

18
Q

True or False:

Children inherit number of STRs from one parent

A

False, children inherit one allele from each parent at a single STR locus

19
Q

How are STRs analyzed?

A

With gel electrophoresis or capillary electrophoresis

20
Q

What is compared in an STR electropherogram?

A
  • Standards and samples
  • Heterozygous vs. Homozygous
21
Q

How is PCR for STR locus done?

A
  1. Primers that will anneal to the sides of the STR are created
  2. Primers will then replicate the section in the middle
  3. Collect the products and run electrophoresis
22
Q

Define:

Electropherogram

A

A graph obtained by capillary electrophoresis

23
Q

What is the difference between capillary and gel electrophoresis?

A

Capillary electrophoresis is the same as gel electrophoresis, just through a tiny tube

24
Q

How is sex determination done with STRs?

A

Looking for AMEL to determine sex
* Shorter in X
* Longer in Y

25
Q

What is one application of PCR?

A

Creating cDNA which is expressable in prokaryotes (bacteria) (e.x. for producing insulin)

26
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats

27
Q

Describe:

The CRISPR locus

A

Has spacers between short palindromic repeats - DNA sequences from previous phage infections (adaptive immune system of bacteria)

28
Q

What is Cas 9?

A

Cas9 is a protein isolated from bacteria

29
Q

Describe:

Phase 1 in CRISPR

A
  1. Cas1 and Cas2 cut viral DNA
  2. Store into CRISPR locus in bacterial genome (spacers between palindromic repeats
30
Q

Describe:

Phase 2 of CRISPR

A
  1. Transcribe CRISPR locus into pre-crRNA
  2. Binds to tracrRNA (hybrid) (process with RNase III)
  3. Binds to Cas9 to create complex
  4. Signals CRISPR-Cas9 to find viral sequence and cut
31
Q

What guides CRISPR to target gene that needs to be edited?

A

Guide RNA/CRISPR-Cas9 hybrid

31
Q

What does Cas9 need beside sequence to be recognized by guide?

A

PAM (protospacer adjacent motif) - NGG - Sequence

32
Q

Define:

NHEJ

A

Non-Homologous End Joining
* Disruption caused so that gene is not functional
* Error prone

33
Q

Define:

HDR

A

Homology Directed Repair
* DNA strand looks for homology, can provide donor DNA with necessary edits (correction or add mutation/gene)
* Not efficient

34
Q

List the 4 components of base editing of a single strand

A
  1. d/nCas: Dead nic, makes single-strand cut
  2. Guide RNA
  3. Cytidine deaminase: Attached to Cas9
  4. Uracil glycosylase Inhibitor (UGI)
35
Q

How CG changed to TA?

A
  1. Cytosine demainase changes C to U
  2. UGI prevents the cell from changing U back to C
  3. Now have a UG, nic to remove G and add UA
  4. Through DNA replication or repair UA changes to TA
36
Q

List

Applications of CRISPR inside and outside the body

A

Inside
* Ocular disease (in vivo)

Outside
* Cancer (designer T-cells)
* Blood disease

37
Q

Why can’t normal T-Cells attack cancer cells?

A

Normal T cells contain PD-1 (protects tissue from autoimmune attack and facilitate tumor progression

My Packs: UWO (BIO 1002) (10 decks)

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