Block 4 Quiz

Question 1

What are restriction enzymes?

Answer 1

restriction endonucleases are enzymes that cleave very specific DNA sequences

Question 2

How many Basepair does restriction endonucleases usually recognize?

Answer 2

usually short (4-8bp)

Question 3

What are palindromes?

Answer 3

they are sequences that read the same 5’ to 3’ on both strands

Question 4

what type of sequences does restriction endonucleases usually recognize?

Answer 4

palindromes

Question 5

What are the two types of DNA libraries?

Answer 5

• genomic DNA libraries

- cDNA libraries

Question 6

How are genomic DNA libraries made?

Answer 6

The entire genome is chopped up using restriction enzymes, cloned into vectors, used to transform bacteria

Question 7

Does each transform bacteria contain the same segment of the genome?

Answer 7

NO

Question 8

what does genomic DNA libraries contain?

Answer 8

contains all sequences in the same genome including coding regions, introns, intervening sequences and promoters

Question 9

What are genomic libraries used for?

Answer 9

To see if something is present or not

Question 10

How are cDNA libraries made?

Answer 10

generated using isolated mRNA from a particular cell or tissue type
mRNA is reverse transcribed by Reverse Transcriptase, and the second strand is synthesized using DNA polymerase
cDNA is ligated into a vector and used to tansform bacteria

Question 11

What do cDNA libraries contain?

Answer 11

contain sequences representing all mRNAs present in the cell or tissue type at the time the mRNA was collected
it only contain mRNA sequences

Question 12

What are cDNA libraries used for?

Answer 12

allow one to see what genes were being expressed in a particular cell or tissue type, and to look at the expression levels

Question 13

what can DNA from cDNA libraries be used for?

Answer 13

can be cloned into expression vector for production of protein

Question 14

What is DNA sequencing?

Answer 14

technique used to determine the exact sequence of a cloned or PCR amplified stretch of DNA

Question 15

Why and how does ddNTP disrupt DNA synthesis?

Answer 15

ddNTP stops the elongation of the DNA after it binds because it lacks the 3’-OH group so phosphodiester bond can’t be formed and elongation stops

Question 16

What are probes used for?

Answer 16

used to identify DNA fragments

Question 17

What are probes?

Answer 17

ssDNA molecule that is labeled using radioactivity

Question 18

What is Hybridization?

Answer 18

1. target DNA is made ss
2. target is immobilized on a solid support so it cannot re-anneal with the original complementary strand
3. ssDNA coated membrane is exposed to probe
   if complementary sequence is present, probe will bind the immobilized ssDNA and can be identifies via Autoradiography

Question 19

How can the immobilized ssDNA be identified during hybridization?

Answer 19

through radiography

Question 20

What are types of probes?

Answer 20

1. Short probes

2. Long probes

Question 21

what are short probes?

Answer 21

chemically synthesized oligonucleotides (2-30 bases)

Same way synthetic primer is made

Question 22

are small probes specific or not?

Answer 22

very specific- it can identify a single base mutation in a sequence

Question 23

How are long probes made?

Answer 23

via one of several molecular biology techniques such as reverse transcription, PCR

Question 24

are long probes specific?

Answer 24

not really. Much less specific than short probes. it can be used to identify similar genes in different organisms or the same gene in different individuals that may not be exactly the same in sequence

Question 25

What is southern blotting used for?

Answer 25

analysis of DNA

Question 26

How is southern blotting done?

Answer 26

- DNA is isolated and subject to restriction digestion - Digested DNA is then subject to get electrophoresis - DNA is denatured, blotted (Immobilized onto a membrane) - Blot membrane is probed

Question 27

What does blotted mean?

Answer 27

DNA is immobilized onto a membrane so it doesn't get reannealed

Question 28

What technique is used to diagnose sickle cell anemia?

Answer 28

ASO Probes

Question 29

What are ASO probes?

Answer 29

allele specific oligonucleotide

Question 30

What is cystic fibrosis caused by? and what does it result in?

Answer 30

caused by a genetic mutation, results in defective CL ion channeling due to improper folding and assembly of CFTR- chloride transport protein prematurely degraded

Question 31

What is CFTR?

Answer 31

cystic fibrosis transmembrane conductance regulator

Question 32

what is a clinical indicative of cystic fibrosis?

Answer 32

elevated chloride levels in sweat, and lack of chloride secretion in the lungs leads to dehydration of the mucus which leads to mucus accumulating in the lungs and obstructing airway passages. also causes pancreatic enzyme insufficiency and poor nutrition among other problems

Question 33

what is the genetic problem behind cystic fibrosis?

Answer 33

3 nucleotide deletion in the CFTR gene that leads to a missing phenylalanine reside

Question 34

How can CF be detected?

Answer 34

using ASO probes

Question 35

What is ELISAs?

Answer 35

Enzyme- linked immunosorbent assays.

Question 36

How does ELISA work?

Answer 36

antigen is bound to the well of a microtiter plate, probed with an antibody linked to an enzyme. detect by adding substrate for enzyme to form a colored reaction

Question 37

is it possible to get a false positive?

Answer 37

yes

Question 38

What do you do if you get a positive on ELISA?

Answer 38

need to do western blot to confirm the positive because it can give false positive. but when its negative, its negative