DNA Tech

Question 1

What is a PCR and its purpose?

Answer 1

Polymerase Chain reaction,

• Used to make multiple copies of a fragment of DNA
• Can be used to identify DNA at a crime scene or what organism the DNA belongs too

Question 2

What are the 3 additional molecules needed to produce copies of the DNA in PCR?

Answer 2

• Deoxyribonucleotides
• Taq polymerase
• DNA Primers

Question 3

Describe the process of PCR?

Answer 3

• The sample of DNA is heated to break the hydrogen bonds and separate the parent strand
• Then the DNA is cooled so that a DNA primer can attach to an end of the DNA
• Once the DNA primer is placed, Taq polymerase places complimentary deoxyribonucleotides to the parent strands to make two complementary strands
• All the strands are heated again to separate the complementary strands from the parent strands and produce a new copy of the DNA,
• This cycle continues and produces copies of the original DNA at a rate of 2^n
• This process can be done rapidly using a thermocycler which regulates the temperature perfectly for PCR to produce many copies of DNA

Question 4

Why is Taq polymerase more efficient to use than a normal DNA polymerase in PCR?

Answer 4

Taq polymerase is resistant to higher heat so will not denature as easily

Question 5

Why must the temperature be lowered again after heating up?

Answer 5

So the primers can form hydrogen bonds with the template parent strand

Question 6

What is the function of Taq polymerase?

Answer 6

Synthesize new strands of DNA, using the DNA primer as a starting point

Question 7

What is the DNA primer’s function?

Answer 7

To act as a starting point for the Taq polymerase

Question 8

What is the purpose of gel electrophoresis?

Answer 8

To separate fragments of DNA

Question 9

What process is used to separate DNA molecules?

Answer 9

Gel electrophoresis

Question 10

What is the method for electrophoresis?

Answer 10

• Start with a tray, filled with an aqueous buffer solution,
• Place a slab of polyacrylamide or agarose gel in the solution with slits for samples of DNA to be added on one side
• Place two electrodes, one anode (+) and one cathode (-) on either end of the agarose gel, the cathode should be on the side of the DNA samples
• Connect the electrodes to a current and since DNA is negatively charged, the DNA will start to repel from the cathode and attract to the anode
• Since the agarose gel is a thick porous solution, the DNA has to weave through pores, the shorter the DNA the further the strand travels in a given time
• This can be used to separate DNA strands efficiently

Question 11

Why is DNA negatively charged?

Answer 11

There is a negative O connected to the P group of the nucleotides

Question 12

What is agarose?

Answer 12

A polysaccharide gel made from seaweed that is used in electrophoresis

Question 13

What is reptation?

Answer 13

The act of squirming along a smooth-walled narrow passage

- How DNA moves through the agarose gell in electrophoresis

Question 14

What is a DNA ladder?

Answer 14

A set of known DNA fragments with different sizes in base pairs
- The sections of cut DNA in electrophoresis

Question 15

What is ampicillin?

Answer 15

A type of antibiotic

Question 16

What does CRISPR stand for and what is its purpose?

Answer 16

Clustered Regularly Interspaced Short Palindromic Repeats

- A method for DNA editing

Question 17

What is E. coli?

Answer 17

Escherichiawhat coli

- A gram-negative bacteria

Question 18

What are gram-negative bacteria?

Answer 18

A bacteria resistant to most available antibiotics

Question 19

What is the general function of guide RNA

Answer 19

Used as a template for when mRNA is edited

Question 20

What is a plasmid?

Answer 20

A small ring of DNA that carries accessory genes separate from the bacteria chromosomes

Question 21

What is bacteria transformation?

Answer 21

When a bacteria is edited by a gene or genes from another strain of bacteria

Question 22

Where was CRISPR found?

Answer 22

The immune system of bacteria

Question 23

What are CRISPRs?

Answer 23

Short repetitive segments of DNA

Question 24

What is a Cas and its function?

Answer 24

CRISPR Associated protein, it breaks a section of DNA while being guided by a segment of DNA

Question 25

Explain the process of using CRISPR to edit DNA?

Answer 25

- Scientists create a guide RNA to match the gene they want to edit - This guide RNA is attached to Cas9 - The Cas9 uses the guide RNA to find a section of DNA and cut it in the host - Once the DNA is cut, uses homology directive repair by using a template of the DNA to help repair the broken segment, this template can be manipulated to add in a different section of DNA and permanently edit the DNA - This can be used to treat genetic diseases and is not limited to humans so is applicable everywhere

Question 26

What is nonhomologous end joining?

Answer 26

Repairing damaged DNA by simpling binding the two broken ends to each other, - This can miss sections of DNA that have been broken off and therefore is prone to mistakes - The resulting gene is normally unusable and turned off

Question 27

What are some issues with CRISPR?

Answer 27

- It is hard to predict the long term effects of CRISPR | - This raises big ethical questions

Question 28

Describe the process of bacteria transformation?

Answer 28

- A restriction enzyme is used to cut a plasmid and a custom section of DNA is inserted in this opening on the plasmid and the DNA is bound into the plasmid using ligation - Then the cell membrane of the bacteria needs to partially break for the plasmid to be taken in, holes are made by cooling down and then heating up the bacteria, - This heat shock will make breaks in the bacteria for the plasmid to enter, the bacteria cells are cooled down quickly after heating to allow the cell membranes to repair though there is a chance the cell dies, - Then the bacteria are cultured to reproduce with the new DNA from the plasmid - The DNA from the plasmid can be utilized to create desired proteins, this could give the bacteria resistance to specific antibiotics or allow it to just mass-produce a protein like insulin

Question 29

Why does there need to be a break in the membrane of the bacteria to insert the plasmid in bacteria transformation?

Answer 29

DNA is negatively charged so the plasmid cannot diffuse through the cell membrane

Question 30

How is the plasmid bacteria prepared to receive the plasmid in bacteria transformation?

Answer 30

Heat shock: - Digest the plasmid - Create a mixture of the bacteria and digested enzymes and cool them down - Then submerge the mixture in a warmer bath and the heat will shock the plasma membrane and break it quickly - Then cool down the mixture so the membrane can be healed - While the membrane was broken, hopefully, the plasmids entered some of the bacteria cells and can be used by the bacteria to produce proteins from its DNA

Question 31

How can you test that the bacteria absorbed the plasmid in bacteria transformation?

Answer 31

- For the first agar culture, place a sample of the bacteria pre-absorption to make sure the bacteria is alive, if there is growth in the agar culture, the bacteria is alive - In the second culture, add the antibiotic and the bacteria pre-absorption to make sure the bacteria is not already resistant to the antibiotic, the bacteria should die and there should be no growth - With your sample of bacteria with the plasmids absorbed, add it to a petri dish to make sure that the bacteria is still alive after heat shock, observe some growth of the bacteria to make sure it's alive - Finally, take a sample of the bacteria after absorbing the plasmid and insert it into a petri dish with the antibiotic, if there is bacteria growth it means some of the cells absorbed the plasmid successfully and developed resistance - There will likely be fewer bacteria in this last Petri dish because there will be some that died from heat shock and some that did not absorb any plasmids.

Question 32

Why can the bacteria die from heat shock?

Answer 32

The cell cannot control what goes in and out with cracks in its membrane, it could easily be invaded by something lethal or lose vital components of the cell