D1.1 DNA Replication

Question 1

What is DNA replication

Answer 1

Process of producing two identical copies of DNA from one DNA molecule.

Question 2

Purposes of DNA replication

Answer 2

Cell division: before cell division, it must double its DNA so that each daughter cell receives a full set of genetic instructions.

Growth and tissue repair: multicellular organisms grow and repair tissue by increasing cell number. Each new cell requires its own copy of the genome.

Reproduction: ensures genetic info is passed on from one gen to next

Question 3

What does semi-conservative DNA replication mean

Answer 3

Semi-conservative: new daughter cells contain one original and one newly synthesized one.

Two strands separate, each one serving as a template for new one to attach.

Question 4

Role of complementary base pairing in DNA replication

Answer 4

Each original strand contains all information necessary to reconstruct new, opposite strand.

A - T
C - G

Ensures new DNA molecules are identical to original.

Question 5

Why do DNA strands have to be separated prior to replication

Answer 5

So that the two strands can act as templates for semiconservative replication.

Question 6

Role of Helicase in DNA replication

Answer 6

Enzyme that unzips and unwinds the parent DNA strands.

Unwinding requires energy from hydrolysis of ATP.

Helicase unwinds helical shape, and then breaks hydrogen bonds between base pairs.

Question 7

Role of DNA polymerases in DNA replication + explain leading and lagging strand

Answer 7

Enzymes that catalyze the synthesis of complementary daughter strands.

Move along template strand by adding free nucleotides based on complementary pairing.

Catalyzes condensation reaction that creates covalent bonds between phosphate group of nucleotide and pentose of other (backbone).

Separate DNA polymerases work on each template strand so the daughter strands are built simultaneously:
one follows direction of helicase (leading strand).
other works in opposite direction (lagging strand)

Question 8

Function of the Polymerase chain reaction (PCR)

Answer 8

Increase specific DNA sequences, creating millions of copies from only a few original DNA molecules.

Question 9

Process of PCR

Answer 9

Denaturation: temperature increased (around 98) to separate DNA strands.

Annealing: temp decreased (60) to allow primers to bind to base pairs on each strand.

Extension: temp increased (72). Heat resistant Taq polymerase replicates DNA to build new DNA strand.

Question 10

Conditions for PCR reaction chamber

Answer 10

Desired DNA section.
Primers that allow replication to occur at desired point.
Taq polymerase.

Question 11

Role of Gel electrophoresis

Answer 11

Separate DNA fragments based on size and charge.

Question 12

Process of Gel electrophoresis

Answer 12

An agarose gel is heated and placed into a tray with wells.

DNA samples mixed with dye are pipetted into the wells.

Gel is placed in electrophoresis chamber that generates a current through the gel.

Negative electrode is placed on side closest to wells. Positive electrode on other side.

DNA has negative charge so will travel towards cathode.

One well contains DNA fragments with known sizes (DNA ladder). Ladder used to determine size of samples.

Question 13

Applications of DNA electrophoresis

Answer 13

DNA profiling for paternity and forensic investigations.

Question 14

Process of DNA profiling

Answer 14

Examining portions of DNA to create a profile unique to the individual.

E.g. crime scene: DNA on cells that are shed in environment sampled.

Genomes contain short, repeated DNA sequences called tandem repeats. PCR used to amplify these repeats, DNA fragments are separated using gel electrophoresis.

Since DNA sequence is the same in all cells of an individual, the evidence DNA must match the suspect DNA in size and number.

Question 15

Examples of sources that can be used for DNA profiling

Answer 15

Blood, Saliva, Hair, Skin cells, Semen (SA), trace DNA.

Question 16

Two functional constraints of DNA polymerase

Answer 16

Can only attach nucleotides to an existing DNA strand: this means that it cannot initiate the replication of a new strand, so RNA primers are used (short sequence of RNA) that provide a starting point for the polymerase to latch onto.

Can only add nucleotides to the 3’ end of an existing nucleotide.

Question 17

Explain the leading and lagging strands of DNA.

Answer 17

New strands can only be assembled by DNA polymerase III in a 5’ to 3’ direction.

Leading strand follows in the same direction as helicase, towards the replication fork, and can continuously attach nucleotides in a 5’ to 3’ direction. (template is 3’ to 5’). Only needs one primer.

Lagging strand created away from the replication fork. A primer is attached to the 5’ to 3’ template strand, and lagging strand works in opposite direction. As helicase continues to unwind DNA, primers attach to new spaces, where DNA polymerase III will attach and add nucleotides in opposite direction until it reaches the previous primer. These fragments are called Okazaki fragments.

Leading = continuous
Lagging = discontinuous

Question 18

Role of DNA polymerase I

Answer 18

Removes RNA primers and replaces it with DNA after DNA polymerase finishes building a segment of DNA.

Question 19

Role of DNA Gyrase

Answer 19

Topoisomerase enzyme that reduces the torsional strain of the DNA double helix.

Question 20

Role of DNA Primase

Answer 20

Attaches primers to DNA template strands to allow DNA polymerase III to latch.

Question 21

Role of DNA Ligase

Answer 21

Creates phosphodiester bonds between the Okazaki fragments (backbone).

Question 22

Process of DNA proofreading

Answer 22

DNA polymerase III adds an incorrect nucleotide, which make a physical distortion.

DNA polymerase III detects it and moves backwards and removes the nucleotide.

Once removed, DNA polymerase III goes back to attaching correct nucleotides.