Chapter 20

Question 1

Define recombinant DNA

Answer 1

Fused DNA molecules from multiple sources

Recombinant DNA technology is fundamental in genetic engineering and biotechnology.

Question 2

Define molecular cloning

Answer 2

Making of recombinant DNA. Has medical implications such as mass insulin production

Molecular cloning involves several steps to insert a gene of interest into a vector.

Question 3

What are the four main steps involved in creating recombinant DNA?

Answer 3

1. The DNA must be cut and transferred out of its source
2. The DNA destination is cut
3. The transferred DNA is put in the destination
4. The DNA is sealed back together

Question 4

What is the function of restriction enzymes?

Answer 4

They initially cut the DNA at specific sequences

Question 5

Define recognition sequence/site

Answer 5

Restriction enzyme’s target sequence

Question 6

What are sticky ends?

Answer 6

Short overhanging pieces of DNA that allow fragments to pair back up with each other

Sticky ends enhance the efficiency of cloning.

Question 7

Define anneal

Answer 7

Interact via base pairing.
Target gene’s and destination DNA’s sticky ends are ligated together by DNA ligase

Question 8

What are blunt ends?

Answer 8

DNA ends with no overhanging complementary pieces

Blunt ends are not as effective for cloning compared to sticky ends.

Question 9

What are the steps for cloning?

Answer 9

1. Cutting target and plasmid with a restriction enzyme
2. Fusing target and plasmid
3. Transform bacteria and select for properly transformed cells
4. Select those that received the target gene

Question 10

What is a plasmid?

Answer 10

A small circular DNA molecule found in bacteria

Plasmids are commonly used as vectors in molecular cloning.

Question 11

What key features does a cloning plasmid have?

Answer 11

• Multiple restriction sites
• Antibiotic resistance gene
• Reporter gene
• Site to prompt plasmid replication

Question 12

What does the antibiotic resistance gene do?

Answer 12

Codes for proteins that break down antibiotics, allowing bacteria to survive antibiotic treatment

This feature is crucial for selecting transformed bacteria.

Question 13

Define transformation (related to plasmid)

Answer 13

Mix bacteria and many plasmid copies together, then shocking the bacteria to make them accept the plasmid.
*Not every bacterial cell will get a plasmid

Question 14

What does multiple restriction sites do?

Answer 14

This makes the plasmid more versatile. Restriction sites are typically found inside the reporter gene, helping confirm successful cloning.

Question 15

What role does the reporter gene play in cloning?

Answer 15

Helps identify which cells received the target gene based on colony color

Commonly, the LacZ gene is used as a reporter.

Question 16

What does the site to prompt plasmid replication do?

Answer 16

Isolate the colonies with the target gene (using their different colors), and prompt this site to express your target gene to mass produce the protein.

Question 17

What is the purpose of PCR?

Answer 17

To rapidly amplify a specific DNA sequence. Important for studying the function of gene.

PCR is essential for cloning or sequencing genomes.

Question 18

What are the main components required for PCR?

Answer 18

• Template DNA
• DNA polymerase
• dNTPs
• Primers

Question 19

How many primers are needed for PCR?

Answer 19

2 primers - complementary to the ends of the target sequence and have a 3’ end for DNA polymerase to add to.

Question 20

What are the three main steps in a PCR cycle?

Answer 20

1) Denaturing of template DNA
2) Anneal primers
3) Extend primers

Question 21

How does the temp change during PCR and why?

Answer 21

1. Increase temp. Causes initial strands to split by breaking hydrogen bonds
2. Lower temp. Primers can now bind to the template
3. DNA polymerase adds dNTPs to make a copy of gene.

Question 22

What is qPCR?

Answer 22

Real-time quantitative PCR that measures gene expression by quantifying specific mRNA levels

qPCR uses fluorescent DNA amplification for measurement.

Question 23

What are the steps to qPCR?

Answer 23

1) Isolate RNA from cells/tissues
2) Reverse transcribe RNA to DNA
3) Perform PCR on target gene
4) Quantify amount of produced DNA

Question 24

What is the main limitation of qPCR?

Answer 24

• mRNA levels do not equal protein levels
• Some RNAs cannot be reverse transcribed

Question 25

What is the difference between qPCR and PCR

Answer 25

qPCR uses DNA-binding strains to measure DNA abundance (such as fluorescence or radiation).

Question 26

What is the purpose of Western Blots?

Answer 26

To measure the abundance of a specific protein

Question 27

What are the steps in a Western Blot?

Answer 27

1. Isolate all proteins from sample 2. Run samples on a PAGE gel 3. Perform a transfer 4. Use an antibody to reveal the protein

Question 28

What is the role of antibodies in Western Blots?

Answer 28

They bind to the protein of interest for detection ## Footnote Antibodies are designed specifically for the target protein.

Question 29

Define SDS-PAGE

Answer 29

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 30

What is Polyacrylamide?

Answer 30

A pore-containing gel that helps separate your protein of interest. Proteins move through the gel based on their size. Bigger proteins move slower.

Question 31

What are the 2 parts to running samples on a PAGE gel?

Answer 31

1. Put the protein mixture in sodium dodecyl sulfate (SDS). This denatures the protein and puts a negative electrical charge on the protein 2. Load denatured proteins into the wells of the gel

Question 32

How do you perform a transfer?

Answer 32

Lay the gel horizontally, place it on a membrane, and apply a voltage to move proteins to the membrane. *Occurs after you have separated your protein from other proteins

Question 33

How do you reveal the protein using antibodies?

Answer 33

Coat the membrane with a liquid full of your antibody. Antibody binds your protein at it's position on the membrane. More protein in initial sample = more antibody that binds Add a chemical that makes the antibody visible. A darker band = more initial protein = greater gene expression

Question 34

What is a Northern Blot used for?

Answer 34

To measure RNA levels for a specific RNA ## Footnote Northern Blots are useful for assessing mRNA abundance.

Question 35

What is the main difference between Northern Blots and Southern Blots?

Answer 35

Northern Blots measure RNA, while Southern Blots measure DNA ## Footnote Both techniques follow similar principles but target different nucleic acids.

Question 36

What are the steps involved in a Northern Blot?

Answer 36

1) Isolate DNA from your sample 2) Run the DNA on a PAGE gel 3) Transfer to a membrane 4) Add a labeled DNA probe to bind target DNA

Question 37

define a probe

Answer 37

DNA molecule complementary to part of your target RNA. Attached to a detectable molecule. After probe binds to your RNA, results are visualized.

Question 38

What method do you use to look at: RNA DNA protein

Answer 38

RNA - qPCR, Northern, FISH DNA - Southern protein - Western, immunostaining

Question 39

What is the difference between qPCR, Northern, and FISH?

Answer 39

qPCR - some RNA can't be reverse transcribed Northern - doesn't require reverse transcription qPCR & northern - measure abundance FISH - RNA detection / where in cell

Question 40

What is FISH?

Answer 40

Fluorescent in situ hybridization. Detects RNA in a cell by using a DNA probe

Question 41

How do you use a DNA probe in FISH?

Answer 41

Design a DNA probe that is complementary to RNA of interest. This probe has fluorescent molecules attached. Add probe to the cell, which binds to target RNA view fluorescence via a microscope.

Question 42

Define immunostaining

Answer 42

Detects protein using an antibody.

Question 43

How does immunostaining use antibodies?

Answer 43

Antibody is conjugated to a fluorescent molecule. This antibody binds to protein of interest when added to the cells, which can be detected through a fluorescent microscope.

Question 44

What is the difference between immunostaining and Western blot?

Answer 44

immunostaining - detects location of protein in cell Western blot - detects abundance of protein

Question 45

Define knockout mice

Answer 45

Mice that have had their specific gene broken in order to study the gene. Break gene by inserting a foreign piece of DNA to disrupt it.

Question 46

How do you make knockout mice?

Answer 46

1. Design foreign DNA piece 2. Add DNA to mouse embryonic stem cells 3. Transfer those cells to pregnant mother 4. Breed mice until you get a knockout line

Question 47

Define a DNA vector

Answer 47

DNA molecule similar to WT gene is added. Vector has an extra sequence in the middle of the gene, disrupting/breakign gene. Vector undergoes homologous recombination with the genome, placing the broken gene in the genome

Question 48

Define blastocyte

Answer 48

Very early phase of an embryo. The stage in which the modified embryonic stem cells are added to. *Causes some of the reproductive cells of the offspring to have the broken gene

Question 49

How do you get the knockout line from the mother?

Answer 49

Offspring of mother containing the broken gene in their reproductive cells are crossed together

Question 50

Define transgenic mice

Answer 50

Mice with an over-expressed gene. Follows same steps as creating knockout mice, but instead use a vector with a more active promoter.

Question 51

Define CRISPR

Answer 51

Clustered Regulatory Interspaced Palindromic Repeats The immune system genes in bacteria

Question 52

Define Cas9

Answer 52

Bacterial endonuclease, which cuts DNA of viruses that enter the bacterium.

Question 53

Define guide RNA

Answer 53

Cas9 binds to this RNA sequence which has a complementary region to a specific DNA sequence (this DNA sequence would be the virus' genome). this RNA brinds it to the DNA sequence, and Cas9 binds and cuts the DNA sequence - destroying the virus

Question 54

How does Cas9 perform gene editing?

Answer 54

1. Cas9 is isolated, and scientists generate their own guide RNA. 2. Cas9 cleaves that DNA. 3. New DNA sequence is floating around, and cell accidentally adds the new sequence

Question 55

How does the cell accidentally add the new sequence during Cas9 gene editing?

Answer 55

Cas9 double-strand breaks, and sometimes the DNA will do nonhomologous end joining. In a subset of the nonhomologous end joining cases, the DNA you are attempting to add gets inserted/ligated in.