Lecture 4 Part 1

Question 1

What are DNA restriction and sequencing?

Answer 1

Fundamental techniques in molecular biology used for precise cutting of DNA and determining nucleotide order.

Question 2

What are restriction enzymes?

Answer 2

Proteins that recognize specific DNA sequences and cut at or near these sites.

Question 3

Where do restriction enzymes naturally occur?

Answer 3

In bacteria as a defense mechanism against foreign DNA.

Question 4

What factors affect the activity of restriction enzymes?

Answer 4

Temperature, presence of cofactors, correct pH, and salt conditions.

Question 5

What is a palindromic sequence in the context of restriction enzymes?

Answer 5

A sequence that reads the same forward and backward on complementary strands.

Question 6

What specific sequence does EcoRI recognize?

Answer 6

GAATTC.

Question 7

What type of ends does EcoRI produce after cutting?

Answer 7

Sticky ends.

Question 8

What is the lottery effect in relation to recognition sites?

Answer 8

The longer the recognition site, the less frequent it will be in a random genome.

Question 9

What percentage of DNA sequences are the same among individuals of a species?

Answer 9

90%.

Question 10

What are polymorphisms?

Answer 10

Individual genetic differences that include differences in restriction enzyme cleavage sites.

Question 11

How can genetic traits be used for identification?

Answer 11

Each variant of polymorphism can be inherited, allowing for personal or parental identification.

Question 12

What are the types of restriction enzymes?

Answer 12

Type I: Cuts at random locations far from recognition sites.
Type II: Cuts at specific recognition sequences.
Type III: Cuts a short distance from the recognition site.
Type IV: Targets modified DNA.

Question 13

What is one application of restriction enzymes in genetic engineering?

Answer 13

To insert or remove genes.

Question 14

What do restriction fragment length polymorphisms (RFLPs) allow for?

Answer 14

Forensic analysis.

Question 15

What is the first step in the restriction digestion process?

Answer 15

Mixing DNA sample with restriction enzyme and buffer containing Mg²⁺.

Question 16

At what temperature are many restriction enzymes incubated for optimal activity?

Answer 16

37°C.

Question 17

What principle does gel electrophoresis rely on?

Answer 17

DNA molecules are negatively charged, allowing migration in an electric field.

Question 18

What happens to DNA samples during gel electrophoresis?

Answer 18

They are loaded into wells of an agarose gel and migrate toward the positive electrode.

Question 19

How do smaller DNA fragments behave in gel electrophoresis compared to larger fragments?

Answer 19

Smaller fragments migrate faster.

Question 20

What is used to visualize DNA in gel electrophoresis?

Answer 20

Ethidium bromide or other dyes under UV light.

Question 21

What is Southern blotting?

Answer 21

A method used to detect specific DNA sequences in a sample.

Question 22

What are the main steps involved in Southern blotting?

Answer 22

Transferring DNA from gel onto a membrane.
Hybridizing with a labeled probe.
Detecting hybridized fragments.

Question 23

What does DNA sequencing determine?

Answer 23

The order of nucleotides (A, T, C, G) in a DNA strand.

Question 24

Who developed the Sanger sequencing method?

Answer 24

Frederick Sanger.

Question 25

What type of technique is the Sanger sequencing method?

Answer 25

Chain termination technique.

Question 26

What is the role of template DNA in Sanger sequencing?

Answer 26

The DNA strand to be sequenced.

Question 27

What is a primer in the context of DNA sequencing?

Answer 27

A short DNA sequence that initiates DNA synthesis.

Question 28

What enzyme is responsible for adding nucleotides in Sanger sequencing?

Answer 28

DNA polymerase.

Question 29

What are dNTPs?

Answer 29

Normal building blocks of DNA (A, T, C, G).

Question 30

What are ddNTPs and their function in Sanger sequencing?

Answer 30

Modified nucleotides that terminate chain elongation.

Question 31

Fill in the blank: The Sanger sequencing method uses _ that lack hydroxyl -OH preventing polymerisation reaction.

Answer 31

ddNTPs.

Question 32

What is the first step in Sanger sequencing?

Answer 32

DNA is denatured into single strands.

Question 33

What happens after primers bind to the template DNA in Sanger sequencing?

Answer 33

DNA polymerase extends the new strand by adding normal nucleotides (dNTPs).

Question 34

What results from incorporating a small amount of ddNTP during Sanger sequencing?

Answer 34

Stopping elongation at a specific base.

Question 35

How are DNA fragments separated in Sanger sequencing?

Answer 35

Capillary electrophoresis.

Question 36

What does a chromatogram display in Sanger sequencing?

Answer 36

Coloured peaks corresponding to the detected nucleotides.

Question 37

True or False: Sanger sequencing processes multiple DNA strands simultaneously.

Answer 37

False.

Question 38

What is a key advantage of Next-Generation Sequencing (NGS)?

Answer 38

Higher throughput (millions of sequences at once).

Question 39

Name one application of DNA sequencing.

Answer 39

Genetic testing, forensic science, evolutionary biology, biotechnology.

Question 40

What role do magnesium ions (Mg²⁺) play in PCR and DNA reactions?

Answer 40

Essential cofactor for DNA polymerase and restriction enzymes.

Question 41

Fill in the blank: In PCR, Mg²⁺ concentration affects _.

Answer 41

Enzyme activity and primer annealing.

Question 42

How does too little magnesium ions (Mg²⁺) affect PCR?

Answer 42

Reduces efficiency.

Question 43

How does too much magnesium ions (Mg²⁺) affect PCR?

Answer 43

Leads to non-specific binding.