D1.1 DNA replication SL

DNA replication

Producing exact copies of DNA with identical base sequences

What is DNA replication needed for in multicellular organisms?

1. Reproduction

2. Growth + tissue replacement

What type of process is DNA replication?

Semi-conservative

Why is DNA replication a semi-conservative process?

Due to complementary base pairings

How is DNA a semi-conservative process?

Each newly synthesized double-stranded DNA molecule contains 1 og strand + 1 newly synthesized strand

• Bc each parent strand acts as a template for DNA replication → determines order of bases → prevents errors

What does the semi-conservative nature of DNA replication + complementary base pairings allow for? and how?

High degree of accuracy in copying base sequences

Why is the semi-conservative nature of DNA replication important? (use slides)

2 important enzymes in DNA replication

1. Helicase

2. DNA polymerase

Helicase

Enzyme that unwinds (DNA double helix) + breaks hydrogen bonds betw DNA strands

DNA polymerase

Enzyme that adds complementary bases / free DNA nucleotides to the template strands → creates new DNA strands (2 full DNA strands)

• Moves along template strand

• Adds free nucleotides to the new strand, creating a bond betw the phosphate of the free nucleotide + the sugar of the last nucleotide on the strand

• Controls addition of free nucleotides to the developing DNA strand

Polymerase chain reaction (PCR)

Amplifies DNA

• Takes small quantity of DNA → copies al the nucleotides → makes millions copies of DNA

• Only amplifies a targeted section of DNA

What is required for polymerase chain reaction?

1. Primers

2. Temp changes

3. Taq polymerase (enzyme)

4. Nucleotides

5. DNA samples

In PCR, what is added to the DNA sample instead of helicase + how?

Heat

• Heat breaks hydrogen. bonds between parent strands

Primer

• Short segment of DNA

• Signals where to start copying

• Complementary to the nucleotides at 1 end of the target DNA to be copied

Taq polymerase

Heat tolerant DNA polymerase enzyme

• Taq = bacteria living in hot springs → heat resistant enzymes

• Heat doesn’t denature the enzyme

What does Taq polymerase do in PCR?

Synthesizes new DNA strands by adding free nucleotides to a single-stranded DNA template

Why does Taq polymerase need to be heat resistant?

Needs to withstand high temps used to denature DNA

• No need to add new enzymes after each cycle

What allows for fast replication in PCR?

Alternating heat (to break H bonds) + cooling (for binding)

3 steps of PCR

1. Denaturation

2. Annealing

3. Elongation

What happens in denaturation in the PCR process?

DNA is heated to separate the strands

• H bonds holding the 2 strands of DNA (backbone) tog break

• So bases are freely exposed

What happens in annealing in the PCR process?

• Sample cooled down

• Primers bind to specific (nucleotide) sequences on each of the single-stranded DNA.

What happens in elongation / extension in the PCR process?

• Sample heated

• Taq polymerase catalyses the building of new DNA strands by extending the primers + adding free nucleotides

• Entire cycle is repeated

How much DNA does each cycle of PCR produce?

Each cycle of PCR doubles the amount of DNA

• 2^n

Gel electrophoresis

Separates DNA fragments

What does gel electrophoresis separate DNA based on?

Separated fragments of DNA according to size

Steps of gel electrophoresis

1. Put fragments of DNA into 1 end of a porous gel

2. Apply electric current

   • Negative electrode must be at DNA end

Why does gel electrophoresis work when an electric current is applied?

1. DNA also negative

2. So negative end of electrode repels DNA

3. Repelling force moves DNA through the gel

Which fragments of DNA move further through the gel?

Shorter fragments

Applications of PCR + gel electrophoresis

DNA profiling for paternity + forensic investigations

Applications of PCR to test for coronavirus

1. Take swab + isolate viral RNA

2. Reverse transcribe. Use RNA to make DNA

3. PCR to amplify DNA + add fluorescent dyes to specific base sequences

Pros + cons of PCR to test for coronavirus

Pros

• Specific, sensitive

Cons

• Expensive, time-consuming

Short tandem repeats

Repeats of a certain sequence of bases

• Diff people have diff no.s of those repeats

How is PCR + gel electrophoresis used for paternity testing?

1. Isolate short tandem repeats (using enzymes)

2. PCR to amplify the sample (of tandem repeats)

3. Separate them using GE

4. GE creates unique banding patterns for each individual

How to identify the parent of a child using banding patterns from gel electrophoresis?

1. Child gets ½ DNA from mom, ½ from dad

2. Ignore pieces of DNA that come from mom

3. Look at pieces of DNA that could come from other parent

4. ½ bands identical with dad, ½ identical with mom

Process of DNA replication

complete

What enhances reliability in experiements>

Increase the no. of measurements in the test

In DNA profiling, how does increasing the number of markers used help?

Reduces the probability of a false match