Cycle 10 Workshop

Question 1

What are the components of PCR?

Answer 1

• Primers
• dNTPs
• Taq polymerase
• MgCl2

Question 2

What does PCR stand for?

Answer 2

Polymerase Chain Reaction

Question 3

List:

The functions of the components of PCR

Answer 3

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

Question 4

What are the steps of PCR?

Answer 4

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

Question 5

Define:

Multiplex PCR

Answer 5

Uses different primers for multiple sequences

Question 6

True or False:

Each step in PCR is timed

Answer 6

True

Question 7

What is PCR based on?

Answer 7

DNA replication

Question 8

What does RT-PCR mean?

Answer 8

Reverse Transcription PCR

Question 9

List:

Steps of RT-PCR

Answer 9

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

Question 10

What is the difference between cDNA and gDNA?

Answer 10

cDNA is produced by RT-PCR and contains no introns

Question 11

What does cDNA stand for?

Answer 11

Complementary DNA

Question 12

True or False:

cDNA only contains expressed genes

Answer 12

True, is does not have introns

Question 13

True or False:

cDNA can exist in our cells

Answer 13

False, cDNA does not exist in our cells

Question 14

What are the 2 main functions of RNase?

Answer 14

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

Question 15

For gDNA:

1. What does it stand for?
2. What does it contain?
3. Where does it exist?
4. How is it obtained?

Answer 15

1. Genomic DNA
2. Contains both introns and exons
3. Exists in the nucleus
4. Obtained using polymerase

Question 16

For cDNA:

1. What does it stand for?
2. What does it contain?
3. Where does it exist?
4. How is it obtained?

Answer 16

1. Complementary DNA
2. Contains only exons
3. Does not exist in human cells
4. Obtained using reverse transcriptase

Question 17

Define:

STRs

Answer 17

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

Question 18

True or False:

Children inherit number of STRs from one parent

Answer 18

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

Question 19

How are STRs analyzed?

Answer 19

With gel electrophoresis or capillary electrophoresis

Question 20

What is compared in an STR electropherogram?

Answer 20

• Standards and samples
• Heterozygous vs. Homozygous

Question 21

How is PCR for STR locus done?

Answer 21

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

Question 22

Define:

Electropherogram

Answer 22

A graph obtained by capillary electrophoresis

Question 23

What is the difference between capillary and gel electrophoresis?

Answer 23

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

Question 24

How is sex determination done with STRs?

Answer 24

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

Question 25

What is one application of PCR?

Answer 25

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

Question 26

What does CRISPR stand for?

Answer 26

Clustered Regularly Interspaced Short Palindromic Repeats

Question 27

# Describe: The CRISPR locus

Answer 27

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

Question 28

What is Cas 9?

Answer 28

Cas9 is a protein isolated from bacteria

Question 29

# Describe: Phase 1 in CRISPR

Answer 29

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

Question 30

# Describe: Phase 2 of CRISPR

Answer 30

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

Question 31

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

Answer 31

Guide RNA/CRISPR-Cas9 hybrid

Question 31

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

Answer 31

PAM (protospacer adjacent motif) - NGG - Sequence

Question 32

# Define: NHEJ

Answer 32

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

Question 33

# Define: HDR

Answer 33

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

Question 34

List the 4 components of base editing of a single strand

Answer 34

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

Question 35

How CG changed to TA?

Answer 35

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

Question 36

# List Applications of CRISPR inside and outside the body

Answer 36

Inside * Ocular disease (in vivo) Outside * Cancer (designer T-cells) * Blood disease

Question 37

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

Answer 37

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