DNA Amplification, Restriction Digest, and Gel Electrophoresis

Question 1

What are the 4 steps of PCR amplification?

Answer 1

1. Add required reagents or mastermix and template to PCR tubes.
2. Mix and centrifuge. …
3. Amplify per thermo cycler and primer parameters.
4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining.

Question 2

What does it mean for DNA to be “denatured” during Step 1 of PCR.

Answer 2

separate the double-stranded template DNA into single strands so that the primers can bind to the target region and initiate extension.

Question 3

What happens during the annealing stage of PCR?

Answer 3

when the temperature is lowered to enable the DNA primers to attach to the template DNA.

Question 4

What is TAQ Polymerase and how does it extend DNA sequences using primers for replication

Answer 4

Once primers are attached, the Taq polymerase takes its position on the strand to produce the new strands by adding the dNTPs.

Question 5

what is produced at the end of PCR amplification.

Answer 5

the specific sequence will be accumulated in billions of copies (amplicons)

Question 6

what is the purpose of PCR amplification.

Answer 6

allows rapid amplification of a specific segment of DNA.

Question 7

what restriction is endonuclease

Answer 7

enzymes that recognize a specific DNA sequence, called a restriction site, and cleave the DNA within or adjacent to that site.

Question 8

what is a restriction site

Answer 8

a sequence of approximately 6–8 base pairs of DNA that binds to a given restriction enzyme.

Question 9

how does a restriction enzyme cut the DNA

Answer 9

When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule.

Question 10

explain the difference between sticky and blunt ends.

Answer 10

-In sticky ends, one strand is longer than the other
-In blunt ends, both strands are of equal length

Question 11

what is digested DNA

Answer 11

Restriction Endonucleases

Question 12

what are Restriction Fragment Length Polymorphisms

Answer 12

differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes.

Question 13

what is a agarose gel

Answer 13

used to separate mixtures of biomacromolecules, such as DNA, RNA and proteins

Question 14

what does it mean to be porus

Answer 14

permeable to outside influences.

Question 15

the setup of an electrophoresis chamber.

Answer 15

1) Pouring the gel,
2) Preparing your samples,
3) Loading the gel,
4) Running the gel (exposing it to an electric field) and
5) Staining the gel.

Question 16

why does DNA move across the gel

Answer 16

when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode.

Question 17

why do bands of DNA move at different speeds to create a unique banding pattern of RFLPs.

Answer 17

Smaller fragments move faster through the gel leaving the larger ones behind and thus the DNA samples are separated into distinct bands on the gel.

Question 18

What is the DNA ladder

Answer 18

Standard that contains fragments of known lengths or concentrations.

Question 19

how do we use the DNA ladder

Answer 19

The bp next to each number in the ladder indicates how many base pairs long the DNA fragment is

Question 20

why does every person have a unique DNA finger print?

Answer 20

Each DNA strand contains a unique sequence or code of genetic information.