Chapter 10.1 Processing DNA ✓

Question 1

What is PCR?

Answer 1

PCR or Polymerase Chain Reaction is a technique used in biology for producing multiple copies of DNA from a sample

Used in DNA fingerprinting and in identifying diseases

Question 2

What does the process of PCR mimic?

Answer 2

It mimics the natural process of DNA replication that happens prior to cell division

Question 3

What are the three steps of PCR?

Answer 3

Denaturing
Annealing
Extension

Question 4

What occurs in the denaturing step of PCR?

Answer 4

-(During natural DNA replication, the enzyme helicase separates the two strands of DNA, allowing each strand to be copied)
-The PCR uses heat to achieve the same function
-Temperatures of around 95°C is used to break the hydrogen bonds holding the two strands together.
-This separates the strands without disrupting each individual strand

Question 5

What occurs in the annealing step of PCR?

Answer 5

-During annealing, temperature is decreased to around 50-60°C
-This allows short strands of DNA called primers to bind to the single DNA strands
-The primers are not random selections of DNA, instead they are complementary to either end of the section of DNA to be copied

Question 6

What occurs in the extension step of PCR?

Answer 6

-The extension step, also called elongation, is similar to what happens during DNA replication.
-In this step, the enzyme DNA polymerase adds new, matching nucleotides to the single-stranded DNA starting from where the primers are attached.
-This process extends the DNA strand, building a new copy.
-However, this new strand doesn’t include the full original DNA sequence; it starts at the primer, not the very end of the DNA.
-Over time, most of the DNA strands created will only be as long as the section between the two primers.

-DNA polymerase can only attach to double-stranded DNA.
-Before the extension step, this only happens where the primers are bound.
-That’s why primers are so important because they provide a starting point for DNA polymerase and kick off the replication process.

Question 7

What is thermocycling?

Answer 7

It is a process of repeated heating and cooling

Question 8

What are primers?

Answer 8

A strand of DNA or RNA that serves as a starting point for DNA replication

Question 9

Which enzyme is responsible for elongation the DNA strand during extension?

Answer 9

DNA polymerase

Question 10

What is taq polymerase and where is it from?

Answer 10

A heat-stable DNA polymerase from the heat-loving bacterium Thermus aquaticus

Question 11

Why is Taq polymerase useful in PCR?

Answer 11

It dosen’t denature at high temperatures and allows PCR to ab automated without adding enzymes after every cycle

Question 12

At what stage of PCR is Taq polymerase most active?

Answer 12

During extension (elongation) phase

Question 13

How has taq polyermase postively impacted the process of PCR?

Answer 13

It has made it more:
-Efficient
-Cost-effective
-Suitable for automation

Question 14

What are restriction enzymes?

Answer 14

An enzyme that cuts strands of DNA at specific sequences of nucleotides

-These enzymes can be used in a range of applications, including DNA analysis
-When restriction enzymes are added to DNA, it cuts the strands into different lengths depending on the base sequence of the specific DNA sample.
-The length of these pieces can be analysed and compared with other DNA samples.

Question 15

What is gel electrophoresis?

Answer 15

It is a technique that is able to separate DNA strands based on their lengths

Question 16

Describe the process of gel electrophoresis*

Answer 16

-The DNA pieces are placed into wells in a semi-solid gel that is immersed in a solution of an electrolyte.
-There are electrodes at either end of the gel
-The negative electrode is closest to the DNA and the positive electrode is at the opposite side.
-As the current is turned on, the smaller DNA pieces move faster than the larger ones and so are located further away from the negative electrode when the current is stopped.
-This results in a pattern of bands that looks similar to the barcodes on products.
-This banding pattern is an individual’s DNA profile, often called a DNA fingerprint

Question 17

What is DNA fingerprint?

Answer 17

A technique that uses the banding patterns of DNA fragments as a means of identification

Also known as DNA profile

Question 18

What happens in the ‘filling the wells’ stage of gel electrophoresis?

Answer 18

-DNA is placed into the well in the gel
-The DNA samples are accurately placed into the wells using a disposable micropipette to avoid contamination across samples.
-When a current is applied, the DNA will move through the gel rather than diffuse through the solution.

Question 19

In gel electrophoresis, where is DNA place in?

Answer 19

In the wells of the gel in the gel electrophoresis machine

Question 20

What is a DNA ladder?

Answer 20

A DNA ladder is a set of DNA fragments of known lengths used as reference to determine the size of the unknown DNA fragments in the sample

Question 21

After the DNA strands have been separated, what methods do scientists use to visualise the DNA?

Answer 21

In short
-Ethidium bromide
-Methylene blue
-DNA probes

-Ethidium bromide can be added to the agar prior to the gel being set so as the DNA moves through the gel, it picks up some of the chemical.
-Then using a ultraviolet light, the DNA is seen
-But Ethidium bromide is a carcinogen and therefore must be handled carefully

-Methylene blue is a dye that binds to DNA
-When the gel is soaked in the dye, the areas containing DNA stain a deeper blue and therefore visible to the naked eye

-Another method is the use of DNA probes.
-There are short sections of a single strand of DNA with a radioactive or fluorescent molecule that binds to the DNA being tested

Question 22

What is DNA sequencing?

Answer 22

The determination of the precise order of nucleotides in a sample of DNA

Question 23

What is nucleotides correctly called?

Answer 23

deoxynucleotide triphosphates

Question 24

What happens when DNA forms?

Answer 24

-Each nucleotide loses two phosphate groups
-The sugar molecule loses a hydrogen atom from the hydroxy group (OH) when it bonds to the phosphate group of an adjacent nucleotide

Question 25

What is dideoxyribonucleotides (ddNTPs)

Answer 25

A modified deoxyribonucleotide that lacks a hydroxyl group of the sugar component

Question 26

What stops elongation and why?

Answer 26

The synthetic nucleotide stops the elongation of the sequence because there is no OH group for the next nucleotide to attach to. ## Footnote -This happens at each of the nucleotides in the DNA sample, creating different lengths of DNA -This can be separted using gel electrophoriesis.

Question 27

What is DNA sequencing used for?

Answer 27

-It can be used to identify mutation or to compare DNA from different organisms -This is useful in identifying inherited disorders such as sickle-cell anaemia, cystic fibrosis and some forms of cancer. -It has also been used for maternity and paternity tests, in cases where the identity of the father or mother of a child is in dispute. -DNA sequencing can be used by scientists to compare species in order to track evolutionary changes

Question 28

What are the four ethical considerations with genetic information?

Answer 28

-Autonomy -Confidentiality -Equity -Privacy

Question 29

What ethical principle is autonomy?

Answer 29

-Respecting the individuals right to make their own decisions, including whether to undergo testing and if tested, their right to access and share the results -It also includes the right of an individual to decide their own future, independent of genetic information

Question 30

What ethical principle is confidentiality?

Answer 30

The use of genetic information is treated sensitively and is accessed only by those who are authorised to access it

Question 31

What ethical principle of equity?

Answer 31

The right to fair and equal treatment regardless of genetic information

Question 32

What ethical principle of privacy?

Answer 32

The right to be 'left alone' and to make decisions regarding genetic testing and the resulting information, independent of others

Question 33

What does PCR stand for?

Answer 33

Polymerase chain reaction

Question 34

What are the three steps in PCR?

Answer 34

-Denaturing -Annealing -Extension

Question 35

Explain the role of a primer in PCR

Answer 35

-A primer is a short, single strand of DNA that attaches to the target sequence. -It shows Taq polymerase where to start and provides a starting point for building the new strand

Question 36

What is the advantage of using taq polymerase over other DNA polymerases?

Answer 36

-Taq polymerase is heat-resistance, so it not destroyed during the high-temperature step that separates DNA strands -This allows the enzyme to keep working through repeated heating and cooling cycles without needing to be replaced

Question 37

Explain the role of restriction enzymes in DNA profiling

Answer 37

-Restriction enzymes cut DNA at specific base sequences -This creates fragments of different lengths, which vary between individuals. -These fragments can then be separated and compared to create a DNA profile

Question 38

Will the shorter or longer lengths of DNA travel the greatest distance during electrophoresis? Explain your answer

Answer 38

The shorter lengths of DNA because the gel acts like a sieve; shorter pieces can move more easily through it, while longer pieces are slowed down

Question 39

Draw a simplified structure of: a) Deoxynucleotide triphosphate b) Dideoxyribonucleotide triphosphate

Answer 39