Topic 2:5a & 5b DNA extraction, PCR and sequencing

Question 1

List some reasons why DNA profiling matters in the real world?

Answer 1

-Paternity (determine the father of a child)

-criminal justice - DNA testing can help solve crimes by comparing the DNA profiles of suspects to offender samples

-siblings or twins - determining relations

-identification - used to identify people that are injured in high risk jobs

Question 2

what is a DNA sample?

Answer 2

an extracted piece of target DNA which can be extracted from a variety of sources, e.g. blood or saliva from a crime scene or a gene of interest from a plant or animal

Question 3

what’s the first step of DNA extraction from a target cell

Answer 3

Cell lysis: Detergents are added to breakdown and dissolve the cell and nuclear membranes to release the DNA.

Question 4

what’s the second step of DNA extraction from a target cell

Answer 4

Protein removal: Protease and RNAase enzymes are added to remove proteins and RNA. The sample is placed into a centrifuge to form a pellet of all the cell debris.

Question 5

what’s the third step of DNA extraction from a target cell

Answer 5

DNA precipitation: Add ice-cold ethanol -> DNA precipitates from solution.

Question 6

what’s the fourth step of DNA extraction from a target cell

Answer 6

DNA purification: The DNA is washed to remove any impurities.

Question 7

what is the purpose of Polymerase Chain Reaction?

Answer 7

Segments of DNA can be multiplied using PCR. Their base sequences can then be identified and analysed.

Question 8

what are the requirements for PCR?

Answer 8

Target DNA sample
Primers
DNA nucleotides
DNA polymerase (thermotolerant)

Question 9

What are primers?

Answer 9

Primers are designed by scientists to identify the location of the target DNA.
They are short segments of single stranded DNA or RNA that can bind to separated DNA strands by complementary base pairing, and initiate replication.

Question 10

what do primers do?

Answer 10

Primers mark the start and finish points for the sections of DNA to be copied.
Primers prevent DNA strands rewinding during a cooling cycle.

Question 11

why are DNA nucleotides required in PCR?

Answer 11

Free floating nucleotides are used to synthesise new polynucleotide strands using the target DNA as a template.
Nucleotides are provided in excess to ensure there are enough.

Question 12

What does DNA polymerase do in PCR?

Answer 12

A thermo-tolerant enzyme (often Taq Polymerase) is used for PCR so that it does not denature in the heating cycles.
DNA polymerase starts at one primer and joins together the positioned nucleotides until the next primer is reached.

Question 13

where is the DNA polymerase for PCR generally obtained from?

Answer 13

A thermo-tolerant enzyme is obtained from Thermophilus bacteria that live naturally in hot waters (Taq polymerase)

Question 14

what are the 4 steps of PCR?

Answer 14

1. Denaturation
2. annealing
3. extension
4. repeat

Question 15

explain the first step of PCR

Answer 15

Denaturation: the temperature is heated to ~ 95°C causing the hydrogen bonds between the polynucleotide strands of the target DNA to break so bases are exposed. (separates strands)

Question 16

explain the second step of PCR

Answer 16

Annealing: the temperature is cooled to ~ 55°C allowing primers to anneal (bind) to the separated strands of target DNA through the formation of hydrogen bonds between complementary bases. The DNA nucleotides also bind to the exposed bases on the template DNA, forming weak hydrogen bonds.

Question 17

explain the third step of PCR

Answer 17

Extension: The temperature is heated to 72°C which is the optimum for DNA polymerase to connect the nucleotides to produce a new DNA strand. (forms covalent bonds between the phosphate-sugar backbone).

Question 18

explain the third step of PCR

Answer 18

Repeat: The PCR cycle of steps 1-3 is repeated approximately 25-30 times to produce sufficient amounts of DNA fragments.

Question 19

describe the nature/rate of amplification of target DNA using PCR

Answer 19

exponential: The total amount of target DNA is doubled every cycle (amplification is exponential) until factors such as primer concentration, enzyme concentration and DNA nucleotide concentration become limiting factors in the reaction.

Question 20

what is the machine that PCR is done in called? What does it contain? What is one use for it?

Answer 20

Thermocycler:
PCR is carried out in small test tubes in a machine called a thermocycler.
It contains positions for multiple test tubes allowing rapid amplification of target molecules in a laboratory setting.
The rapid nature of PCR is useful in medical diagnosis where doctors amplify DNA taken from human stool to test for the presence of pathogenic bacteria.

Question 21

PCR is one way of obtaining many copies of a target DNA strand. What’s another?

Answer 21

Mitochondria also contain small circles of DNA called mitochondrial DNA (mtDNA).
Each cell contains hundreds of mitochondria so when investigating the genes in mtDNA, PCR is not used to amplify DNA but the mtDNA is simply extracted from mitochondria as sufficient mtDNA is obtained this way

Question 22

The base sequence of DNA can be observed by:

Answer 22

electrophoresis

Question 23

DNA sequencing can be used to construct…

Answer 23

DNA profiles. They may be displayed in an electropherogram or in a table of data

Question 24

What is Gel electrophoresis used for in general?

Answer 24

Gel electrophoresis is a technique used to separate DNA molecules/fragments of different sizes.

Question 25

Describe the set up of electrophoresis

Answer 25

Samples of DNA are loaded into wells on a plate of agarose gel that is attached to two electrodes; positive (+) and negative (-). A battery or power supply maintains a constant electric potential difference between the two electrodes.

Question 26

Why does DNA migrate across the agarose gel?

Answer 26

DNA fragments are negatively charged due to the presence of phosphate groups in the polynucleotide strands and are attracted to the positive electrodes.

Question 27

Why Does separation of DNA occur during electrophoresis?

Answer 27

Separation of DNA fragments occurs due to the molecules of DNA migrating through the agarose gel at different rates. Smaller molecules travel at a greater rate and move a greater distance from the negative electrode (starting point). Larger molecules travel at a slower rate and move a shorter distance from the negative electrode.

Question 28

describe what a marker DNA strand is, and what it is used for in gel- electrophoresis

Answer 28

A separate marker DNA is also loaded onto the gel. The marker DNA consists of a series of fragments of known size. The marker fragments are used to determine the size of the fragments in the sample by comparison.

Question 29

What is the step after DNA separation has occurred in gel- electrophoresis

Answer 29

A fluorescent dye is added to the DNA after separation has occurred. The dye bonds with DNA and causes the molecules to fluoresce under ultraviolet (UV) radiation.

Question 30

What is the last step in gel- electrophoresis that allows for analysis?

Answer 30

An image called a DNA profile is taken which allows an analyst to identify and analyse the different sized DNA fragments in the sample.

Question 31

State what DNA sequencing is

Answer 31

DNA sequencing is the process of determining the nucleotide base sequence of a target DNA molecule.

Question 32

What is one method of DNA sequencing?

Answer 32

The chain termination method. This involves carrying out PCR using modified DNA nucleotides called dideoxynucleotides (ddNTPs).

Question 33

DNA sequencing involves two processes:

Answer 33

electrophoresis, and a DNA sequencing technique, often the chain termination method.

Question 34

what is a dNTP?

Answer 34

a dNTP is a deoxynucleotide. (i.e just a regular nucleotide). There are 4 types for ATCG: dATP, dTTP, dCTP, dGTP

Question 35

what is a ddNTP?

Answer 35

a ddNTP is a dideoxynucleotide. These have a slightly different structure, so can be added to a growing DNA molecule but then cannot form sugar-phosphate bonds with other dNTPs or ddNTPs. So a ddNTP is always the last nucleotide of a fragment as it causes termination of the growing polynucleotide strand in DNA replication and PCR. There are 4 types for ATCG: ddATP, ddTTP, ddCTP, ddGTP

Question 36

Why are ddNTPs used in PCR?

Answer 36

The chain terminating property of ddNTPs makes them suitable for DNA sequencing

Question 37

state the 7 steps of DNA sequencing, by the chain termination method

Answer 37

1. Four test tubes are each filled with many copies of the target DNA molecules to be sequenced. 2. All four deoxynucleotides (dNTPs) are also added to each test tube (A,T,C,G) in excess. 3. Many copies of the one type of dideoxynucleotide (ddNTP) is added to each test tube, so each test tube has a different type of ddNTP (but many copies of it). 4. Primers and DNA polymerase enzymes are added to each of the four test tubes. 5. The four test tubes are transferred to a thermocycler where DNA replication occurs 6. DNA replication is stopped whenever a ddNTP is added instead of an dNTP. This method produces many incomplete (chain terminated) copies of the template DNA, each ending in a ddNTP. 7. the contents of the test tubes are transferred to the agarose gel, and electrophoresis occurs

Question 38

electrophoresis through a capillary tube: explain the fluorescent colours

Answer 38

A fluorescent chemical compound is attached to the ddNTPs used in DNA sequencing, and both are positioned at the end of the DNA fragment. A different fluorescent colour indicates a different ddNTP positioned at the end of a DNA fragment.

Question 39

electrophoresis through a capillary tube: explain the process of separation

Answer 39

Gel electrophoresis is used to separate the fluorescently labelled DNA fragments by size. This process takes place in capillary tubes inside a DNA sequencer. DNA fragments migrate through the capillary tubes at different rates with smaller fragments moving at a greater rate than larger fragments.

Question 40

electrophoresis through a capillary tube: how is light emission important?

Answer 40

All fragments pass through a beam of laser light which is absorbed by the fluorescent molecule attached to the ddNTP. The fluorescent molecule emits a light which enters a detector. The detector converts the light into an electric current that is analysed by computer software.

Question 41

What does the computer software do with the light that is detected by the laser beam?

Answer 41

The detector converts the light into an electric current that is analysed by computer software. The software produces a graph of the data received from a DNA sequencing machine. The graph is called an electropherogram.

Question 42

What is an electropherogram?

Answer 42

A graph that shows the DNA sequence of a target DNA molecule that has undergone electrophoresis in a DNA sequencer. The height of each peak represents the amount of light absorbed and emitted by the fluorescent molecule attached to the ddNTP at the ends of a DNA fragment. The order of the peaks represents the nucleotide sequence of the target DNA molecule.

Question 43

The information in an electropherogram can be used to determine:

Answer 43

The order of base sequences of unknown segments of DNA. Changes in the base sequence of specific segments (or genes) electropherograms of different genomes which may identify presence of a genetic disease. Identify genetic differences between individuals within and between species. This could be for disease detection, prevention, and diagnosis, forsensics or family history or paternity. Evolutionary relationships between different species.

Question 44

state why DNA can be obtained from faeces

Answer 44

Faeces contain blood cells as well as epithelial cells from the stomach, smallintestine and large intestine which contain DNA from the koala.

Question 45

state 2 properties of PCR that make it suitable for identification of pathogenic bacteria

Answer 45

PCR is fast/rapid which allows rapid diagnosis of infectious disease before symptoms become worse. PCR is highly sensitive; only a small amount/sample of DNA is required to obtain results.

Question 46

Describe how PCR and DNA profiling are used to diagnose infectious disease caused by pathogenic bacteria

Answer 46

PCR is used to amplify the DNA from the stool sample of a patient: DNA profile is produced using the amplified DNA from PCR: The DNA profile is compared with a DNA profile of the pathogenic bacteria to determine if the bacterium is present.