Bacterial Genetics

Question 1

DNA is a molecule made up of units called what?

Answer 1

nucleotides

Question 2

Briefly identify the three parts of a nucleotide.

Answer 2

Nucleotides have 3 parts,
a deoxyribose sugar with
a phosphate connected to one end,
and one of four nitrogen-containing bases on the other side of the deoxyribose sugar.

Question 3

Name the four nitrogenous bases of DNA.

Answer 3

(adenine, thymine, cytosine, guanine)

Question 4

Briefly describe how nucleotides form a DNA molecule.

Answer 4

These are linked together to form a chain that connects to an opposite chain, forming a double-stranded DNA molecule.

Question 5

Briefly describe how nitrogenous bases link up to give DNA its shape.

Answer 5

The nitrogen-containing bases form hydrogen bonds with a counterpart base on the opposite chain forming the classic double helix structure of DNA.

Question 6

We take advantage of what characteristic of DNA to work with DNA in the laboratory?

Answer 6

the specific base pairing for each base, copies of DNA are made by using one strand as a template to create a new, opposite strand.

Question 7

The sequence of nitrogen-containing bases in the DNA provides what for the function of a cell?

Answer 7

the information to create specific products, like enzymes, that carry out cell metabolism.

Question 8

Briefly describe mutations and their consequences.

Answer 8

Changes in these sequences are mutations that may or may not change how the information is processed.

Question 9

What technique is used to acquire enough DNA for analysis?

Answer 9

To acquire enough DNA for analysis, the PCR technique is used.

Question 10

What is required to start the PCR technique?

Answer 10

To start, a template DNA strand is needed. Before amplification with PCR, DNA must first be isolated from the cells in a patient or environmental sample.

Question 11

Describe the Several key materials needed for PCR.

Answer 11

Samples for PCR are prepared in labeled microcentrifuge tubes by adding DNA samples along with a master mix containing the heat-stable polymerase, deoxynucleotides, primers, and polymerase buffer.

Question 12

Give an example of heat-stable polymerase and describe its function.

Answer 12

A heat stable polymerase such as Taq polymerase builds the new DNA molecules from free deoxynucleotides added to the reaction.

Question 13

Explain why a heat-stable polymerase is required and briefly describe its source.

Answer 13

The process is carried out at high temperatures so a heat stable polymerase is used, generally taken from thermophilic (heat loving/heat thriving) bacteria.

Question 14

Briefly describe primers needed for PCR and their function.

Answer 14

primers, short DNA fragments with a known sequence used to direct the polymerase to specific areas on the template DNA strand that flank the target DNA sequence.

Question 15

Name the machine used for PCR and its purpose.

Answer 15

Prepared samples are placed in a thermocycler, a machine that repeats three temperature changes needed for each PCR cycle.

Question 16

Name the steps of the PCR cycle.

Answer 16

The PCR cycle has 3 steps:
* Denaturation - Heating to 95°C is used to break the hydrogen bonds between nitrogenous bases and separate the strands of DNA.
* Priming – After cooling, the primers attach to their complementary sites on the separated DNA strands.
* Extension – The sample is warmed back up to 72°C and the polymerase begins synthesizing new strands starting at the primers.
These steps are repeated over and over till the desired amount of DNA is synthesized.

Question 17

Briefly describe the denaturation step of the PCR cycle.

Answer 17

Denaturation - Heating to 95°C is used to break the hydrogen bonds between nitrogenous bases and separate the strands of DNA.

Question 18

Briefly describe the priming step of the PCR cycle.

Answer 18

Priming – After cooling, the primers attach to their complementary sites on the separated DNA strands.

Question 19

Briefly describe the extension step of the PCR cycle.

Answer 19

Extension – The sample is warmed back up to 72°C and the polymerase begins synthesizing new strands starting at the primers.

Question 20

Briefly describe the usefulness of gel electrophoresis to PCR.

Answer 20

The products of PCR are detected by using gel electrophoresis. Many copies of the target DNA are produced which can be separated by size in a gel. These are compared to positive control or a DNA ladder to determine if the specific target fragment is present.

Question 21

Describe what PCR gel electrophoresis entails.

Answer 21

Gel electrophoresis entails:
* Preparing the DNA samples
* Using an electrophoresis gel and chamber to separate the fragments
* Using a staining process to visualize the gel results after sample fragments are separated by electrophoresis

Question 22

Describe biotechnology.

Answer 22

the use of living organisms and their biological processes to make useful products and address practical problems.

Question 23

Describe how DNA is isolated from a sample for use with PCR.

Answer 23

This involves breaking down cell membrane structures to release the DNA from the cell. The extraction process uses enzymes to digest these structures. The sample is centrifuged to collect all the cell debris at the bottom of the tube, leaving the DNA suspended in the fluid above.

Question 24

In the labs, the DNA is already isolated, so what steps does the student perform to begin the PCR process?

Answer 24

The PCR process starts with adding DNA samples to a tube and then adding the master mix solution.

Question 25

Describe the purpose of the loading dye to the PCR process.

Answer 25

The loading dye contains the indicator bromophenol blue to visualize where the samples are located in the gel

Question 26

Describe the purpose of glycerol to the PCR process.

Answer 26

glycerol which helps the sample sink to the bottom of the well and not float on top of the buffer solution.

Question 27

Describe electrophoresis chamber assembly.

Answer 27

• first placing the casted gel onto the platform of the chamber partially filled with buffer solution.
• The chamber has positive and negative electrodes at opposite ends, and the gel is oriented with the wells closest to the negative electrode.
• The chamber is then filled with buffer solution to conduct the electrical current.

Question 28

Describe the step of loading the gel.

Answer 28

• The DNA samples are loaded into the wells at one end of the gel with a micropipette that measures volume in microliters.
• New disposable tips are used for each sample to prevent cross contamination.
• Each sample is carefully placed into the correct well in the gel so the sample does not disperse into the buffer solution and no air is introduced into the well.

Question 29

Describe the purpose of including a DNA ladder made up of a collection of DNA fragments of known length.

Answer 29

One well contains a DNA ladder made up of a collection of DNA fragments of known length. As these spread through the gel, they provide a reference point to any sample fragments that line up in the same location. This allows for the estimation of the length of a given sample fragment.

Question 30

Describe the step of running the gel.

Answer 30

• Finally a lid is placed on the chamber and the electrical current is turned on.
• The negative DNA fragments will migrate through the gel towards the positive electrode. Small fragments move more quickly, and larger ones more slowly through the gel.
• The DNA fragments can migrate off the far end of the gel if power is left on too long. The loading dye makes the location obvious so it can be prevented.
• While the gel runs, fragments are separated by size, which forms a pattern of DNA fragments in the gel matrix.
• Each unique DNA sample will form a different pattern.

Question 31

Describe the purpose of a stain to analyzing the gel.

Answer 31

To see where the DNA fragments are in the gel, the samples all contain a stain, like ethidium bromide, which binds to DNA molecules for detection and allows visualization of the DNA bands in the gel.

Question 32

Name the stain mentioned in the lab used to visualize the DNA fragments in a gel.

Answer 32

ethidium bromide

Question 33

Briefly describe DNA ladder.

Answer 33

Solution of DNA fragments of known size used to compare unknown DNA to estimate fragment size.

Question 34

Briefly describe dNTP mix.

Answer 34

Deoxynucleotides needed for the PCR process.

Question 35

Briefly describe electrophoresis chamber.

Answer 35

Buffer-filled box where a separation gel is placed, and a current is applied to separate DNA fragments by size.

Question 36

Briefly describe ethidium bromide.

Answer 36

A molecule that binds to DNA bases and fluoresces under UV illumination to visually detect the location of DNA bands in the electrophoresis gel.

Question 37

Briefly describe SYBR Green I.

Answer 37

A safer alternative to Ethidium Bromide used to visually detect the location of DNA bands in the electrophoresis gel.

Question 38

Briefly describe loading dye.

Answer 38

aka indicator dye
A solution added to an electrophoresis sample to give it color and density.

Question 39

Briefly describe the master mix of the PCR process.

Answer 39

Contains Taq polymerase, primers, deoxynucleotides, and polymerase buffer solution.

Question 40

Briefly describe microcentrifuge.

Answer 40

Small tabletop centrifuge that uses centripetal force to separate substances by density.

Question 41

Briefly describe microcentrifuge tube.

Answer 41

Small tube with a lid holding less than 2 mL used in a microcentrifuge.

Question 42

Briefly describe micropipette tip.

Answer 42

Disposable plastic tip holding different volumes of liquids.

Question 43

Briefly describe restriction enzyme.

Answer 43

An enzyme that cuts DNA at specific recognition sites called restriction sites.

Question 44

Name two example restriction enzymes from the sim labs.

Answer 44

EcoRI and Psti enzymes are examples.

Question 45

Briefly describe Taq polymerase.

Answer 45

A DNA polymerase enzyme adapted to working at high temperatures without denaturing.

Question 46

Briefly describe 10x Taq buffer.

Answer 46

A concentrated solution used to maintain conditions suitable for the activity of the Taq DNA polymerase.