D1.1

Question 1

DNA Replication

What is DNA Replication?

Answer 1

• The production of new strands of DNA with identical base sequences to existing strangs.
• Involves separating the double strands of DNA then using each strand as a template to copy and new complemenary strand across from it
• Involves copying the entire genome

Question 2

DNA Replication

What is meant by Semi-conservative?

Answer 2

• At end of DNA replication there’s two double stranded DNA helicase, each contains old strand of old DNA and newly made strand built through replication
• This production of ‘half-old, half-new’ is semi-conservative replication.

Question 3

DNA Replication

What are the two processes requiring DNA replication

Both processes, occurs to make new cells.

Answer 3

• Growth and Repaid of Tissue. DNA is replicated before cell division by mitosis.
• Reproduction. DNA is replicated before meiosis, which produces sex cells.

Question 4

DNA Replication

What is the role of complementary base pairing?

Answer 4

• Because of the specific base pairing rules each newly separated single strand provides the information for what should be across from it
• Every strand of DNA is the template/instructions for its complementary strand.
• Faciliates semi-conservation replication

Question 5

DNA Replication

What is the significane of the Messelsohn-Stahl Experiment?

Answer 5

• What’s originally clear whether replication was semi-conservative or conservative
• DNA is scattered through BOTH new strangs
• Experiment used nitrogen isotopes to track original DNA through generations to provide empirical support that replication is SEMI-conservative

Question 6

DNA Replication

What is the role of Helicase?

Answer 6

• Helicase is a ring shaped protein/enzyme that separates the two original DNA strands by breaking the weak H-bonds between strands.
• Ring shape faciliates this by pulling one strand through the ring, so as ring moves along, it quickly breaks H-bonds to pull apart bonds.

Question 7

DNA Replication

What is the role of DNA polymerase III?

Answer 7

• Group of enzymes that attach a new DNA base to the growing complementary strand being assembled (5’–>3’ on both lagging and leading strands)
• There exists free nucleotides in the nucleus, so DNA polymerase uses base pairing rules to add the correct nucleotide
• H-bonds cause it to attach to template & DNA polymerase catalyses the formation of phosphodiester backbone bond to add it to growing new strand.
• Also proofreads new strand and removes non-complementary placements.

Question 8

DNA Replication

What is a Replicaiton fork?

Answer 8

• Place where double stranded DNA is split into two single strands.
• Place where helicase is currently working.
• Moves as helicase unzips more of the DNA
• Note that there’s two replication forks (replicaiton moves in both directions to copy entire chromosome)

Question 9

DNA Replication

What is a primer?

(RNA primer)

Answer 9

• New strand of DNA requires a small fragment of RNA to initiate new strand
• DNA polymerase III extends strands by adding bases to existing string but cant initaite a new strand
• Hence, RNA primers must be firstly placed down to act as the starting point for the new strand.
  Leading: ONE RNA primer - Lagging: multiple required.

Question 10

DNA Replication

What is the LEADING strand?

Answer 10

• DNA polymerase III can only add bases to a free 3’ end of a nucleotide, thus must build strands in the 5’–>3’ direction.
• Leading strand can be built continously, and is produced more rapidly.

Question 11

DNA Replication

What is the LAGGING strand?

Answer 11

• Also necessary to build a 3’–>5’ (upside down) strand across from original 5’–>3’
• More difficult, involves building small fragments backwards and then combing them together afterward.
• More time consuming, thus lags behind.

Question 12

DNA Replication

What are Okazarki Fragments?

Answer 12

• The fragments in the lagging strand due to it being built backwards to maintain a 5’–>3’ direction of replication.
• They are short-copied framents
• They’re eventually glued together to create one continuos strand of DNA.

Question 13

DNA Replication

What is DNA proofreading?

Answer 13

• Ensuring that the correct complementary base is added during DNA replication.
• This reduces occurance of mutations
• In prokaryotes it is done by DNA polymerase III which can cut out the mistaken base.

Question 14

DNA Replication

What are two limitations of DNA polymerase?

Answer 14

• Cannot initate a new strand: must add to an existing nucleotide which requires primers.
• Can only add a new base to the 3’ end of an existing nucleotide, so can only build a 5’–>3’ (lagging strand dilemma)

Question 15

DNA Replication

Explain the directionality of DNA polymerase and the lagging strand.

Answer 15

• Because DNA polymerase III can’t add a nucelotide onto the 5’ prime end of an existing strand, it can’t use the first primer placed across from the original 5’–>3’ strand.
• It thus requires a second primer that has to be placed further down
• Then a strand can be built 5’–>3’ between two primers (backwards)

Question 16

DNA Replication

What does DNA primase do in replication?

Note the focus is on specific enzymes used by PROKARYOTES

Answer 16

• DNA primase is the enzyme responsible for building & placing correct complementary RNA primers across from the original DNA strands to initate replicaitno.
• One for leading strand, many required for the lagging strand.

Question 17

DNA Replication

What does DNA polyermase I do in replication?

Lagging strand, prokaryotes

Answer 17

• On the lagging strand, multiple primers used.
• After DNA Okazarki framents are built between them, it’s important to remove the RNA primers and replace them with DNA
• This is done by DNA polymerase I, as it can both remove the RNA primers and add/conntecting new DNA nucleotides
• It can’t seal the gap between that primer and the next okazarki fragment

Question 18

DNA Replication

What does DNA ligase do in replication?

The last enzyme needed in prokaryotes to complete the lagging strand

Answer 18

• After DNA Poly I has replcaed the RNA primers with DNA, ligase creates phosphodiester bonds between Okazarki fragments
• Thus, these fragments appear as one continous DNA strand (same as leading strand)

Question 19

PCR and Gel Electrophoresis

What is thermocycler?

Answer 19

• Small machine that can run multiple PCR samples at the same time.
• Involves metal wells that can be rapidly heated and colled
• Polymerase Chain Reaction works by rapid temp changes that faciliate the steps needed to rapidly copy DNA
• Thermocycler quickly alternates the 3 different temps to allow one round of PCR after the next = quick succession.

Question 20

PCR and Gel Electrophoresis

What are primers in the context of PCR?

Answer 20

• Like in DNA replication, needed to start the new strands of DNA being built but are instead made of DNA
• Crucial for PCR as often only one sequence of DNA is to be copied and the primers bind to the beginning and the end of the target sequence of DNA
• Means they both identify the DNA to be copied & start need strands for polymerase to act on.

Question 21

PCR and Gel Electrophoresis

What is Taq polyermase?

And what is its role in PCR?

Answer 21

• Used because it is not denatured by heat used to split the double stranded DNA.
• Can retain structure even in extreme temps and functions optimally at 72 degrees in the final stages of PCR
• It adds complementary bases to the strand started by primers.

Question 22

PCR and Gel Electrophoresis

What is Gel electrophoresis?

Answer 22

• Tools that allows negative DNA fragments to move through a porous agarose gel towards a positive electrode
• The current is then stopped
• Smaller DNA fragments move more rapidly than large DNA fragments, so the fragments are scattered through the gel by size, creating a barcode unique to people (DNA fingerprint)

Question 23

PCR and Gel Electrophoresis

What are short tandem repeats?

Electrophoresis

Answer 23

• Short, 2-7 base pair fragments that are repeated
• DNA fingerprints/profiling by eletrophoresis benefits from using these sections of DNA that are usually variable

Question 24

PCR and Gel Electrophoresis

What is DNA profiling?

Answer 24

• The proccess of creating a unique DNA banding pattern using restriction enzymes that cut DNA (often short tandem repeats) and gel electrophoresis that then separates those fragments

Question 25

# PCR and Gel Electrophoresis What is the purpose of PCR?

Answer 25

* To take **small amounts of DNA** and create **many copties**, often specific fragments of interst. * Allows for a small sample of DNA to generate large amounts of target DNA, that produces a visable strands on gel after electrophoresis.

Question 26

# PCR and Gel Electrophoresis Explain the stage of denaturation in PCR.

Answer 26

* The double stranded DNA is **separated into two single strands** * Done with heat that breaks the **weaker H-bonds** * Done at **95** degrees for 30-60 seconds

Question 27

# PCR and Gel Electrophoresis Explain the stage of annealing in PCR.

Answer 27

* After DNA is separated, tempearture **drops to 54** degrees and the added primers bind to the target sequences of the separated strands. * Shows **polymerase** where to copy and starts the strands.

Question 28

# PCR and Gel Electrophoresis Explain the stage of extension in PCR.

Answer 28

* The final stage, at **72 degress** celcius as this is the optimum temperature for **taq polymerase** * Taq adds the **complementary free nucleotides** to the primers to build new complementary strands *This creates **2 strands**. All 3 steps repeated 25-30 times.*

Question 29

# PCR and Gel Electrophoresis Explain separation by size by electrophoresis.

Answer 29

* **DNA has a negative charge** so when connected to power, is attracted to the positive electrode. * The argarose gel has small pores that DNA moves through * **Small DNA** fragments can move more **rapidly** to the **positive electrode**. * **Larger DNA** move more **slowly** *This separates by size*

Question 30

# PCR and Gel Electrophoresis Explain Electrophoresis for Paternity Testing.

Answer 30

* **DNA samples from child, mother & POTENTIAL fathers** * Short tandem repeats are often used (no. repeats is inherited and variable) * Child should have the **same no. repeats as mum/dad** * Bands most of interst are those a child has that their **mum does not**, meaning it must come from dad.

Question 31

# PCR and Gel electrophoresis Explain PCR for testing for COVID-19

Answer 31

* From swab, the enzyme **reverse transcriptase** converts viral RNA to DNA. * Specific primers attach to this DNA to copy fragments only found in the virus. * **Flurorescent markers** are added which bind to the **viral DNA** is presented * Once PCR has created enough of the viral DNA, fragments are visable.