D1.1 DNA Replication

Replica

Exact copy of something

DNA replication

Production of new DNA strands with base sequences which are identical to existing strands

Reasons for DNA replication

• Reproduction

• Growth

• Tissue replacement

Replication fork

Site where DNA replication is actively occuring

State how new strands are formed

By adding nucleotides one by one and linking them together

State why DNA replication is referred to as semi-conservative

Presence of two DNA molecules, both consist of

• An original strand

• A newly synthesised strand

Template strand

Original DNA strand that acts as a “guide” for the replication of new ones

Explain what happens when the wrong base sequence is inserted

• No hydrogen bonding between bases

• Nucleotide would be rejected

Replisomes

Assemblages of functional subunits

State why DNA strands must be separated prior to replication

So each strand can serve as a template for the new DNA strands

Role of helicase in DNA replication

Unwinding and breaking of hydrogen bonds between DNA strands

Briefly explain how helicase works

• One strand is pulled through ring hole

• Other is passed to the side of it

• DNA is uncoiled

General role of DNA polymerase

Assembles new strands of DNA, using the two original strands as templates

Briefly explain how DNA polymerase works

Adds one nucleotide on strand

PCR

Polymerase chain reaction:

• Doubles the quantity of DNA with each cycle

Process of PCR

• Denaturing of DNA/RNA

• Annealing, temp decreases

• Elongating

• Amplifying

State why PCR cycle is known as a thermal cycle

Because its steps are triggered by temperature

DNA amplification

Producing more DNA with a specific base sequence

Primer

Short single strand of DNA that binds to the point of DNA where selected base sequence for replication begins

Taq polymerase

Heat-tolerant DNA polymerase- in charge of DNA amplification

Gel electrophoresis

Process by which DNA is separated by length

Process of gel electrophoresis

• Put DNA fragments into one end of porous gel

• Apply electricity (negative electrode at DNA end)

• DNA is also negative, causing repulsion of DNA through the gel

Explain how PCR and gel electrophoresis are used in testing for coronaviruses

• Throat/nasal swab is taken

• Virus particles and viral RNA are rinsed off in saline solution to produce liquid sample

• RNA is converted to DNA using reverse transcription

• PCR is used amplify specific viral base sequences (about 35 cycles of PCR are used)

• As PCR progresses flourescent markers are attached to any DNA produced, fluorescence leves are monitored, if the go above target, result of test is positive

Explain how PCR and gel electrophoresis are used in testing for paternity tests

• DNA sample is obtained

• Selected tandem repeats are copied by PCR

• DNA produced by PCR is separated according to length of fragment and therefore number of repeats using gel electrophoresis

• Pattern of DNA bands is produced on the gel

  • For paternity testings → DNA profiles of child, mother and father in question are compared

Explain/Describe the structure of a nucleotide

• Deoxyribose (DNA) or oxyribose (RNA)

• 5’ and 3’ ends where nucleotides are received

Outline how DNA polymerase assembles new DNA strands

By linking together a strand of nucleotides with bases complementary to those of the template strand

5’ terminal vs 3’ terminal

• 5’: site available for linkage is on fifth carbon

• 3’: site available for linkage is on the third carbon

State the direction in which DNA polymerase always is in

5’ of a DNA nucleotide is added to the 3’ end

Distinguish between replication on the leading and lagging strand

Leading strand	Lagging strand
DNA polymerase adds nucleotides moving towards replication fork	DNA polymerase adds nucleotides moving away from the replication fork
Replication is continuous	Replication is restarted/discontinuous
Replication is completed more quickly	Replication is relatively slow
RNA primer initiates replication only once	RNA primer initiates replication repeatedly

Okazaki fragments

Short lengths of new DNA strands

Function of DNA primare

Lays down an RNA primer to provide a binding site for DNA polymerase III

Role of DNA polymerase III

Adds new nucleotides in the 5’ to 3’ direction

Role of DNA polymerase I

Removes all the RNA primers and replaces it with correct DNA nucleotides

Role of DNA ligase

Connects Okazaki fragments and nucleotides laid down by DNA polymerase I and II

DNA proofreading

• Done by DNA polymerase II

  • Removes any nucleotides from 3’ terminal

  • Replaces mismatches with correctly matched nucleotide

What should be made at the end of DNA replication?

2 identical DNA strands