Unit 3 Exam lecture 18

Question 1

Restriction enzymes do what

Answer 1

recognize specific sequences and cut the DNA at defined sites

Question 2

restriction enzymes leave what two types of ends

Answer 2

blunt or sticky ends

Question 3

HindII leaves what kind of ends

Answer 3

sticky

Question 4

PvuII leaves what kind of ends

Answer 4

blunt

Question 5

How do we find and isolate a gene of interest

Answer 5

using restriction enzymes

Question 6

How to we separate the gene of interest that we want to study

Answer 6

use of souther blotting or restriction enzymes to isolate

Question 7

What is the overall process of southern blotting

Answer 7

1. digest genome with restriction enzyme
2. separate on gel
3. use a complementary probe to isolate the gene of interest
4. autoradiography to detect fragments on probes

Question 8

Southern blotting detects what
Northern blotting detects what
Western blotting detects what

Answer 8

1. DNA
2. RNA
3. Protein

Question 9

what is the caveat of southern blotting

Answer 9

need to have probe that is complementary otherwise will not be able to isolate

Question 10

What are the three things that you need in a cloning vector to insert a genome

Answer 10

1. origin of replication recognized in host cell
2. selectable markers
3. single cleavage site for each restrictive enzyme used

Question 11

What is the process of inserting foreign DNA into the plasmid

Answer 11

1. plasmid and foreign DNA are cut by same restriction enzyme
2. when mixed sticky ends anneal
3. Nicks sealed by DNA ligase

Question 12

How is the plasmid with foreign DNA placed into the cell

Answer 12

transformation

Question 13

what are the effects of inserting foreign DNA into the middle of the lacZ gene

Answer 13

when grown on special gel, bacteria with recombinant plasmid will not synthesize B-galactosidase and their colonies will be white while the other will be blue

Question 14

benefit of including a marker with antibiotic resistance

Answer 14

only cells which take up the plasmid will be able to survive and proliferate

Question 15

What are the steps in PCR

Answer 15

1. Heating to separate
2. Annealing of primers
3. Elongation

repeat

Question 16

What are the limitations of PCR

Answer 16

need primers that are specific to the sequence you want to proliferate, primers could anneal to the wrong DNA

Question 17

What was the original way that DNA was sequenced

Answer 17

Used ddNTPs which lack 3’ OH group,
four reactions each with a different base ddNTP,
all have the 4 deoxyribonucleotide triphosphates,
when ddNTP incorperated into genome chain stops,
get different lengthed fragments,
run 4 reaction results through a gel to determine complementary sequence to the original strand.

Question 18

Describe the innovation and design of the Sanger Sequencing method

Answer 18

single stranded DNA fragment
four ddNTPs tagged with different flourescent dyes
primer added and chain extended
products denatured and fragments run in gel
DNA is detected by laser beam to determine order of sequence
computer takes data and converts it to target sequence

Question 19

What is the process of Illumina sequencing

Answer 19

Place entire genome
restriction enzymes cut it up
add specific addaptors for one primer to fragments
single primer type added
primer binds to adaptor
only need one primer and adaptor for everything

Question 20

What is a contig

Answer 20

alignment of DNA fragments at the points where they overlap to make consensus contig (real sequence of genome)

Question 21

Forward genetics are what

Answer 21

starting with phenotype and proceeding to genotype

Question 22

Reverse genetics are what

Answer 22

starting with genotype and altering the sequence to determine effects on phenotype

Question 23

What is site directed mutagenesis

Answer 23

creation of transgenic animals by adding sequence of interest to genome of organisms that normally lacks sequence

Question 24

what is benefit to using mammals for site directed mutagenesis

Answer 24

oocytes are large enough that DNA can be injected directly

Question 25

Explain the process of creating transgenic mice

Answer 25

Euthinized males have sex with females to get hormones going but no sperm foreign DNA injected into pronuclei embryos implanted in pseudopregnant female offspring are tested for presence of transgene

Question 26

What is the success rate of creating transgenic organisms

Answer 26

low, 10-30% of eggs survive and most do not have copy

Question 27

What is the point of inserting foreign DNA early in development when creating a transgenic animal

Answer 27

so most if not all copies of chromosomes have the foreign DNA

Question 28

What does the target sequence of DNA need for CRISPR-Cas9 to work

Answer 28

complementation with the sgRNA and need for PAM

Question 29

What happens to the DNA after it is cleaved by Cas9

Answer 29

fragments will either join together or their will be new donor DNA inserted

Question 30

Why would it be better to change Cas9 to make single stranded breaks

Answer 30

easier to insert new DNA than double stranded breaks

Question 31

When should genes be inserted or destroyed while using Cas9

Answer 31

Early in development or end up with mosaics

Question 32

What are the limitations to CRISPR-Cas9

Answer 32

Off target cleavage, sgRNA doesnt have to match 100%

Question 33

siRNAs do what

Answer 33

switch off gene expression

Question 34

siRNAs need to be recognized and cleaved by

Answer 34

Dicer

Question 35

Why can siRNAs not be used on gene families

Answer 35

homology is too similar and siRNA will not be specific enough

Question 36

ApoB is a key part of what

Answer 36

lipoproteins

Question 37

genetic mutations that cause elevated levels of ApoB cause

Answer 37

increased chance of coronary artery disease

Question 38

How to potentially combat elevated levels of ApoB

Answer 38

RNAi has been demonstrated to be able to reduce levels of ApoB

Question 39

What is one reason that using synthesized siRNAs can be difficult (evident in monkey study)

Answer 39

need to find way to get them into cell

Question 40

How were scientists able to get ApoB-siRNAs into primate cells

Answer 40

encapsulated ApoB-siRNAs in lipids