09b: Modern Techniques

Question 1

To isolate genomic DNA for analysis, which cell(s) are used for sample?

Answer 1

Lymphocytes

Question 2

To isolate mRNA for analysis, which cell(s) are used for sample?

Answer 2

The target tissue

Question 3

To isolate proteins for analysis, which cell(s) are used for sample?

Answer 3

The target tissue

Question 4

Genomic DNA is different in which specific tissue type(s)?

Answer 4

SAME IN ALL CELLS

Question 5

Genomic DNA is used for analysis to investigate:

Answer 5

1. Genetic mutations
2. Gene promoter sequences
3. Exon/intron junctions

Question 6

Genomic DNA cannot provide information about:

Answer 6

Any tissue-specific expression

Question 7

T/F: Fully processed mRNA is the only type of mRNA that can be isolated and analyzed.

Answer 7

True

Question 8

mRNA is used for analysis to investigate:

Answer 8

1. Tissue-specific gene expression levels
2. Expression level differences in normal and tumor cells
3. Different developmental stages
4. Transcriptional/post-transcriptional modification

Question 9

mRNA cannot provide information about:

Answer 9

Gene structure

Question 10

Proteins are used for analysis to investigate:

Answer 10

Post-translational processing (phosphorylation, proteolytic cleavage, etc)

Question 11

All sequences cleaved by restriction enzymes are:

Answer 11

DNA palindromes

Question 12

(X) are bacterial enzymes that have evolved as protection mechanism (protect from invading organisms).

Answer 12

X = restriction enzymes

Question 13

Restriction enzymes can generate which types of ends?

Answer 13

1. Blunt

2. Sticky (3’ or 5’ overhangs)

Question 14

(Blunt/sticky) ends are more precise.

Answer 14

Sticky

Question 15

In Southern blot, the blot contains (X) and the probe is (Y).

Answer 15

X = DNA
Y = DNA

Question 16

In Northern blot, the blot contains (X) and the probe is (Y)

Answer 16

X = RNA
Y = DNA

Question 17

In Western blot, the blot contains (X) and the probe is (Y).

Answer 17

X = protein
Y = antibody

Question 18

In (X) blots, the probe is (single/double)-stranded DNA of (known/unknown) sequence.

Answer 18

X = Southern and northern

single-stranded, known sequence

Question 19

List ways in which a Northern blot probe can be generated.

Answer 19

ssDNA probe can be generated via:

1. Synthesis
2. Restriction fragment
3. PCR

Question 20

Couple wants to know if they’re carriers of sickle cell mutation. You take a sample from which tissue(s)?

Answer 20

Lymphocytes (to isolate genomic DNA)

Question 21

How does the probe stick to the sample (DNA/RNA/protein) on a Southern blot?

Answer 21

DNA; the DNA probe is ssDNA and the DNA on the blot is denatured, ssDNA as well. Complementary binding of probe to target sequence

Question 22

You’re using (X) blot to check for specific disease mutation. Which sequence do you put on the probe?

Answer 22

X = Southern

ssDNA containing the mutation

Question 23

In blot, which tool is used to see the potential adherence of probe to sample?

Answer 23

Probe is labeled with radioactive (32)P before incubated with blot; then expose to X-RAY

Question 24

What’s RFLP? What is it used for?

Answer 24

Restriction Fragment Length Polymorphism; used to identify mutations which affect restriction sites

Question 25

The (X) mutation in sickle cell disease impacts (Y) site. Thus, a normal sequence will differ in mutated sequence in which way?

Answer 25

``` X = point (Glu to Val) Y = restriction ``` Normal sequence will be cleaved, but SS sequence will not be cleaved

Question 26

How does the probe differ in RFLP and in southern blot?

Answer 26

It doesn't differ

Question 27

Could you tell a SS carrier apart from SS homozygous patient via RFLP?

Answer 27

Yes - one allele differs between them, so the carrier would have one mutated and one normal (total of 3 bands instead of one on gel)

Question 28

The incredible thing about PCR is:

Answer 28

Ability to replicate DNA in quick cycle, due to heat-stable Taq Polymerase

Question 29

How is PCR used in forensics?

Answer 29

Make primers for the universal "flanking regions" of STR sites

Question 30

How does CRISPR work?

Answer 30

1. Virus invades bacteria 2. "Spacer" sequence derived from virus and integrated into CRISPR sequence 3. CRISPR RNA foremed 4. CRISPR RNA guides Cas9 nuclease to target and destroy viral genome

Question 31

What's cDNA?

Answer 31

Copy of DNA that's complementary to specific RNA sequence

Question 32

(X) can be used to produce cDNA from (Y).

Answer 32

``` X = reverse transcriptase Y = RNA sample ```