D1.1: DNA Replication

Question 1

State what DNA replication is

Answer 1

The production of exact copies of DNA with identical base sequences.

Question 2

Outline the purposes of DNA replication.​

Answer 2

• to provide a complete copy of the organism’s DNA so that the organism grows from a fertilised egg cell to a large multicellular organism
• repairing damaged tissues

Question 3

Describe the meaning of “semi conservative” in relation to DNA replication.

Answer 3

the products of DNA replication each contain one of the original DNA strands and one new strand

Question 4

Explain the role of complementary base pairing in DNA replication.

Answer 4

The complimentary base pairing rule ensures the new strands built are exact copies of the original & genetic code remains intact between generations
- A binds with T
- C binds with G

Question 5

State why DNA strands must be separated prior to replication.

Answer 5

• each strand of the original DNA molecule acts as a template for the new DNA strands that are being built

Question 6

Outline the role of helicase in DNA replication.

Answer 6

Helicase is an enzyme attaching to DNA
- It moves along the molecule unwinding the DNA helix and separating the 2 strands by breaking H bonds betw. the bases

Question 7

Outline the role of DNA polymerases in DNA replication.

Answer 7

• DNA Polymerase III moves along template strand from 3’ to 5’ and assembles new strands of DNA in the 5’ to 3’ direction. Replicates continuously on the leading strand, replicates lagging strand discontinuously.
• DNA Polymerase I removes RNA nucleotides of the RNA primers and replaces them with the correct DNA nucleotides

Question 8

State the arrangement of DNA polymerase and template DNA for each strand

Answer 8

There are separate DNA polymerases for each stand of template DNA.

Question 9

State the function of the PCR.

Answer 9

Amplifies a specific DNA sequence, producing multiple copies of it

Question 10

Outline the process of the PCR, including the use of primers, temperature changes and Taq polymerase.

Answer 10

denaturation phase;
- DNA heated at around 98*C to break the H bonds that hold the 2 strands of the double helix tgt

annealing phase (cooling);
- The sample cools at around 60*C and the short RNA primer sequences bond to complementary sequences in the DNA sample.

extension phase;
- The bonding of RNA primers allows Taq Polymerase to replicate DNA using the RNA primer was a starting point at around 72*C; the DNA polymerases are only able yo extend existing strands and not able to add the first nucleotide of a DNA strand. DNA strands are heated and the process repeats

Process is repeated and the region of interest is amplified exponentially

Question 11

Deduce the number and relative size of DNA fragments from the number of bands in an electrophoresis gel.

Answer 11

250 to 30k base pairs

Question 12

Outline the procedure for DNA electrophoresis.

Answer 12

• Gel electrophoresis uses electrical currents to move molecules through a gel
• DNA separated by size and amount of charge
• DNA fragments get digested using restriction endonuclease enzymes which cut the backbone of DNA double helix making shorter DNA segments
• samples of DNA fragments loaded into wells on one end of the gel which is submerged in a buffer solution
• DNA samples begin near negative pole so that they can spread out as they are drawn towards positive pole because PO4 3- group of DNA is negatively charged

Question 13

Describe how and why DNA fragments separate during electrophoresis.​ ​

Answer 13

• samples of DNA fragments loaded into wells on one end of the gel which is submerged in a buffer solution
• DNA samples begin near negative pole so that they can spread out as they are drawn towards positive pole

Question 14

List applications of the PCR.

Answer 14

• DNA profiling
• Cloning of genes
• Identification of bodies
• To sequence the DNA of extincr species and other life forms

Question 15

Outline the use of the PCR in testing for viral infection.

Answer 15

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Question 16

Discuss advantages and disadvantages of using the PCR test for viral infections.

Answer 16

advantages:
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disadvantages:
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Question 17

Outline the process of DNA profiling.

Answer 17

• There are short repeated DNA sequences called tandem repeats
• Amplification with PCR
• The resulting mix of DNA fragments gets separated using gel electrophoresis

Question 18

List applications of DNA profiling.

Answer 18

Unique patterns of bands can be used:

• in forensics, can compare the pattern from a crime scene to patterns of victims/suspects
• in paternity testing, pattern of an individual is compared with patterns of possible parents

Question 19

Analyze a DNA profile to determine relatedness or forensic guilt.

Answer 19

sharing of a unique feature

Question 20

List example sources of DNA that can be used in DNA profiling.​

Answer 20

• residue on a doorknob
• a drinking glass
• a piece of clothing

Question 21

Identify the 5’ ends and 3’ ends of a strand of DNA.

Answer 21

• 5’ end is the line connecting deoxyribose sugar and phosphate group
• 3’ is counted clockwise from O at top of deoxyribose

Question 22

Describe the formation of the covalent bond between adjacent nucleotides during DNA replication.

Answer 22

• These phosphodiester bonds occur between the phosphate group attached to the 5ʹ carbon of the deoxyribose of one nucleotide and the hydroxyl group on the 3ʹ carbon of deoxyribose on the next nucleotide

Question 23

State what DNA polymerases can only add to an existing DNA strand.

Answer 23

DNA polymerases can only add free nucleotides to an existing DNA strand.

Question 24

State what DNA polymerases can only add the 5’ phosphate of a free nucleotide to of the elongating strand.

Answer 24

DNA polymerases can only add the 5’ phosphate of a free nucleotide to the 3’ deoxyribose of the elongating strand.

Question 25

Explain why replication is different on the leading and lagging strands of DNA.

Answer 25

Leading Strand: - DNA Polymerase III moves along template strand in 3' to 5' direction and new strand is synthesised continuously in 5' to 3' direction Lagging Strand: - DNA Polymerase III moves along template strand in 3' to 5' direction and new strand is synthesised discontinuously in 5' to 3' direction hence, creating Okazaki fragments which are short fragments of DNA. - Ligase joins Okazaki fragments together by catalysing the formation of a phosphodiester bond

Question 26

Compare the pace and direction of replication on the leading and lagging strands of DNA.

Answer 26

Leading strands of DNA (similar to the sense strand as it mirrors the sequence of the RNA produced) : - faster pace - Direction of replication is 5' to 3' Lagging strands of DNA (similar to the antisense strand as it serves as a template for building a new strand): - slower pace - Direction of replication is 5' to 3'

Question 27

Outline the formation of Okazaki fragments on the lagging strand.

Answer 27

- discontinuous replication occurs on the lagging strand - DNA polymerase replicates the new strand in sections having to repeatedly move further along the strand to continue replicating it - These sections of newly formed but short and disconnected DNA are Okazaki fragments that are joined on the lagging strand by DNA Ligase

Question 28

Explain the need for RNA primers in DNA replication.

Answer 28

RNA primers allow DNA Polymerase to attach and begin assembling the free nucleotides into a new strand of DNA - without the RNA primers, DNA polymerase is unable to attach to the DNA strand properly

Question 29

Compare the number of RNA primers on the leading and lagging strands.

Answer 29

- a single RNA primer is required on the leading strand as replication is continuous - primers need to be placed at regular intervals on the lagging strand to allow DNA polymerase to attach at multiple points necessary for discontinuous replication that occurs on this strand

Question 30

Outline the function of the enzyme DNA primase.

Answer 30

Attaches RNA primers made of RNA nucleotides to the template strand - This allows DNA polymerase to attach and begin assembling the free nucleotides into a new strand of DNA

Question 31

Outline the function of the enzyme DNA polymerase III.

Answer 31

- Assembles new strands of DNA by placing free nucleotides in the correct sequence according to the base sequence of the template strand and the complementary base pairing rule - Replicates continuously on leading strand but must replicate lagging strand discontinuously meaning it must replicate the strands in short sections called Okazaki fragments

Question 32

Outline the function of the enzyme DNA polymerase I.

Answer 32

Removes RNA nucleotides of the RNA primers and replaces them with the correct DNA nucleotides

Question 33

Explain why there are gaps between adjacent Okazaki fragments on the lagging strand.

Answer 33

- Discontinuous replication that occurs in the lagging strand as DNA polymerase III can only add nucleotides in the 5' to 3' direction

Question 34

Outline the function of the enzyme DNA ligase.

Answer 34

Catalyses the formation of the phosphodiester bonds between the Okazaki fragments. This makes the replicated strand built using the lagging strand into a single strand that can function normally.

Question 35

State the function of DNA proofreading.

Answer 35

corrects errors during replication.

Question 36

Outline the process of DNA proofreading by DNA polymerase III.

Answer 36

- DNA PIII adds an incorrect nucleotide whilst building the new strand of DNA (due to mispairing) - DNA PIII identifies the error of the mismatched base at the 3' end of the mismatched strand - DNA PIII splits phosphodiester bond between mismatched nucleotide - DNA PIII removes incorrect nucleotide - DNA PIII adds correct nucleotide