Molecular Lab Pre-Lab Quiz 2

DNA = ?

Deoxyribonucleic acid

What does DNA contain?

The information for the development and functioning of all living organisms

What type of strands are found in DNA?

Polynucleotide

What bonds the 2 strands of DNA?

Hydrogen bonds

How do the 2 DNA strands run?

Antiparallel

In what direction are DNA sequences read?

5' to 3' direction

Why is DNA read from 5' to 3'?

Because enzymes can only add new nucleotides to a free 3′ hydroxyl group (-OH), while the template strand is read in the opposite 3′ to 5′ direction

How many bonds are between A and T

2 hydrogen bonds

How many bonds are between C and G

3 hydrogen bonds

Where is DNA located in plant cells?

- Nucleus

- Mitochondria

- Chloroplasts

What part of a plant is best for isolating DNA?

Tissues

What type of plant tissue is best for isolating DNA?

Young tissues

What (physical) plant parts are used to isolate DNA

- Leaves

- Roots

If you don't immediately process the DNA of a plant you've isolated, what should you do?

- Freeze it

- Store it appropriately

What is the homogenization of tissues?

The mechanical process of breaking down solid biological samples into a uniform, evenly mixed liquid or suspension

If you break down a cell's wall, does that make DNA extraction take more or less time?

More time

PCR =

Polymerase Chain Reaction

What does PCR do?

- Amplifies DNA

- Acts like a Xerox machine for DNA

What technique was used before PCR became commonplace?

Cloning

What is cloning and what are its downsides?

- Cloning makes copies of DNA

- Takes a very long time

- Requires a very high quality source of DNA

What are the advantages of PCR?

- It is speedy, we can create millions of copies (of DNA) in hours

- It can use small samples of DNA, or even degraded samples

Is PCR easy or cheap?

Yes, it's both easy AND cheap

Why is PCR like the body?

The process mimics what your cells do on their own

What process does PCR mimic specifically?

In vivo replication

Step 1 of in-vivo replication

DNA unwinds

Step 2 of in-vivo replication

Expose single-stranded areas of DNA to be copied (ssDNA)

Step 3 of in-vivo replication

Primer is introduced and used to initiate replication

Step 4 of in-vivo replication

DNA polymerase adds nucleotide base pairs to make the complementary strand (based on the ssDNA template)

How is PCR different from DNA Replication?

They use the same ingredients and methods, but PCR uses temperature changes (heat) to complete the process

—> DNA replication does NOT require heat

What are the 3 steps to PCR?

1) Denaturation

2) Annealing

3) Extension

At what temperature is denaturation (for PCR testing) carried out at?

- 94 deg Celsius (very hot)

What happens during denaturation?

- We are breaking DNA apart

- Heat melts the double-strand of DNA into ssDNA (single-stranded)

- It does so by breaking the hydrogen bonds which bind base pairs

At what temperature is Primer Annealing carried out at for PCR?

- Between 45 and 60 deg Celsius

How does temperature change from Step 1 of PCR to Step 2?

We go from boiling to a cooler temperature

What happens during primer annealing?

The mixture is cooled so that primers can bind to their complementary DNA strands via hydrogen bonds

What does a primer do?

They tell us where/what pieces of DNA we're copying

What type of sugar is a primer?

Oligosaccharide

When we're amplifying a region of DNA, what 2 components are the ends of the strand?

- Forward primer

- Reverse primer

What happens in Extension (Step 3) during a PCR?

- Taq Poly adds nucleotides to the attached primer, forming a new complementary strand

- We're building a nice, long piece of complementary DNA

At what temperature is extension carried out? Is that hot?

- 70 deg celsius

- Hot, but not so much that it denatures the DNA

In what direction does extension occur?

Extends in a 5' to 3' direction

What is the most common polymerase?

DNA Polymerase

Optimal temperature for DNA Poly

37 deg C

Denaturation temperature for DNA Poly

50 deg C

Are PCR temperatures compatible for normal polymerase activity?

No --> PCR works at a temperature of 94 deg C which is far too high

Taq = ?

Thermus aquaticus

Where does Taq polymerase come from?

It is created from a bacterium that lives in hot springs

What is Taq polymerase called?

"Thermostable enzyme"

Why is Taq polymerase our polymerase of choice? (for PCR testing)

- It is thermostable

- Its optimal activity range falls within the temperatures that PCR functions at

Optimal activity range of temps for Taq polymerase

50 deg C to 80 deg C

Up to what temp can Taq polymerase withstand/work?

Up to 95 deg C

What is the co-factor for Taq polymerase?

Magnesium Chloride

What is a reaction buffer used for, and what does it contain?

- It's used to maintain pH

- And it contains salt

If we use Taq polymerase over and over again, what can we achieve?

Exponential Amplification

What is exponential amplification?

When PCR is repeated over and over, and the region of interest (DNA strand) is amplified exponentially

--> AKA we get an exponential amount of DNA as a result

With a successful PCR, how many bands should you see per well?

1 band (clear, sharp)

Which is stronger/more accurate--PCR or ELISA?

PCR

What advantage does ELISA testing have over PCR testing?

- PCR testing cannot be done at home

- ELISA testing CAN be done at home, making it that much efficient and accessible

--> ELISA doesn't require the extra, complex equipment that PCR does

What is a GMO?

A genetically modified organism

Genetically modified crops usually contain what, and this achieves what effect?

- Contain foreign genetic material (usually a gene)

- This gives an advantage to that plant

What is Step 1 of producing a GMO?

Identifying the potentially beneficial gene--knowing that gene we're trying to work with/isolate, why we're doing that

What is Step 2 of producing a GMO?

Isolating and cloning the gene using a plasmid

What is Step 3 of producing a GMO?

Engineering the gene

If you are isolating a gene and inserting it into another organism, what regions of DNA are important?

- Coding

- Non-coding (too)

Are non-coding regions necessary and/or valuable?

Yes, extremely

When preparing a gene for GMO, what does the coding region represent?

The coding region codes for the protein you want your GMO to make, hence why you're isolating it

What is the promoter region?

A specific sequence of DNA bases at the start of a gene where DNA polymerase binds

What is the terminator region?

A specific sequence of DNA bases marking the end of the transcription process (tells DNA Poly to stop transcribing)

If you insert solely the coding region of a gene into an organism, what happens?

- Nothing happens

- The organism views the strand as trash

Why are non-coding regions essential to GMO?

When inserting the gene of interest, the non-coding regions help orient the organism to understand that what's being inserted is useful

—> And they provide instructions for cellular machinery

Why are promoter regions from plant viruses used in GMOs?

Because plant viruses infect plant cells, their promoter regions can function in plant cells, allowing the inserted gene to be expressed throughout the plant.

What type of molecule is CaMV 35S

Promoter

What is CaMV 35S derived from?

It's from the Cauliflower Mosaic Virus

Is CaMV 35S widely useful?

Yes, it functions in every plant cell

What type of molecule is NOS?

Terminator

What does NOS stand for

nopaline synthase

What is NOS derived from?

Soil bacterium Agrobacterium tumefacians

What is a selectable marker, and what is it used for in genetic engineering?

selectable marker (e.g., GFP or antibiotic resistance) is used to identify which cells contain the engineered gene.

What are some good GMO modifications that can be made?

- Drought-resistance

- Pest-resistance

- Improvement in nutritional value

What is a potential downside to GMOs?

There is the risk of the GMO changing something within their growing environment

What is Golden Rice, and why is it considered a beneficial use of GMOs?

Golden Rice is a genetically modified version of rice--it was GMO'd to include the missing component needed for Vitamin A synthesis--now causing the rice to produce β-carotene (provitamin A).

--> This has helped reduce Vitamin A deficiency and childhood blindness in 3rd world countries

DNase

An enzyme that cuts DNA

Why is DNase found all over our skin?

We naturally produce lots of DNase, which is found on our skin

--> It helps protect us from incoming viruses and pathogens.

How does the InstaGene Matrix protect your DNA sample from DNase?

InstaGene mix chelates (binds) to Mg²⁺, removing Mg²⁺ from the sample

--> Without Mg²⁺ as a cofactor, DNase (enzyme) cannot function properly, so it doesn't break down your DNA sample.

3 Pros of GMOs

1) Reduce the use of pesticides/herbicides

2) Reduce stress to the land

3) Improve food's nutritional value

3 Cons of GMOs

1) Expensive

2) Can create antibiotic resistance

3) Can cause allergic reactions