Biology - D1.1 DNA replication

Definition DNA replication

Production of exact copies of DNA with identical base sequences

Why is DNA replication necessary

For reproduction and growth and tissue replacement in multicellular organisms (before mitosis and meioses)

Positive aspect of complementary base pairing, role in DNA replication

The strand that unwinds and acts as a template has bases attach that are complementary so DNA replication is accurate and reliable, few mistakes but can check pairing and replace bc know complementary base pairing

Semi-conservative meaning and application

Each new DNA molecules is made of an original strand and a newly synthesised strand

Half of the original has been conserved

Definition replisome

Proteins (some of which are enzymes) that work together in DNA replication

List of enzymes involved in prokaryotic DNA replication

1) Helicase

2) DNA primase

3) DNA polymerase III

4) DNA polymerase I

5) DNA ligase

What does helicase do?

• Unwinds the coiled DNA

• Breaks hydrogen bonds between complementary base pairs

• Thus, separating original DNA strands, so that one is ready to act as template strand

• Works at the replication fork

What’s the replication fork?

Where the double strands of DNA split and where helicase works

SL: What does DNA polymerase do?

• Adding the correct nucleotides through complementary base pairing

• Forms new hydrogen bonds between base pairs

• Forms covalent bonds between phosphate and sugar

• Works on both strands

Directionality of DNA replication

Nucleotides can only be added in 5’ (5 prime) to 3’ direction

Therefore new nucleotides add at the 3’ end of previous nucleotides

2 different strands due to directionality

• Leading strand (nucleotides added continuously)

• Lagging strand (nucleotides added discontinuously, in Okazaki fragments)

What does DNA primase do?

• Complementary nucleotides have to be added after primer

• so DNA primase synthesises short segment of RNA and adds it (once for leading strand, and every 100-200 bases for lagging strand)

What’s a primer?

• Short segment of RNA (10 nucleotides long)

• Joins to DNA temporarily

• Enables DNA polymerase to bind and start replication

• On leading strand there’s only 1 needed, on lagging strand its every 100-200 bases

What does DNA polymerase III do?

• Adds nucleotides in 5’ to 3’ direction

• Makes hydrogen bonds

• Makes covalent bonds between phosphate and sugar

• Proofreads each nucleotide and corrects any mistakes

DNA polymerase I function DNA replication

• Removes RNA primer and replaces it with DNA (may leave a slight gap between DNA from DNA polymerase and new DNA)

DNA ligase function in DNA replication

• Seals the gaps where the RNA primer has been removed

• Makes covalent bonds between adjacent nucleotides

What enzyme does DNA proofreading?

DNA polymerase III, removes any nucleotides from 3’ terminal that has a mismatched base

Definition PCR

Polymerase chain reaction

An automated and artificial method of replicating DNA, to amplify DNA

DNA amplification definition

To make more copies of a particular sample of DNA

What is targeted to replicate?

A specific section fo DNA, usually the sections that vary considerably between individuals

The short tandem repeats (STRs) where the amount of times the non-coding region repeats varies from person to person. 13 of these.

Targeted with specific primers

What are the stages of PCR

Denaturation, annealing and elongation

How much DNA is replicated in one cycle

DNA is doubled, and one cycle takes 2 minutes

In 1 hr there are 30 cycles and DNA is amplified by factor of a billion

What is put into machine at the start?

DNA, DNA nucleotides, primers and taq DNA polymerase

What happens in the first step?

Denaturation

• Sample heated to 95°C to break the hydrogen bonds and separate the two strands

What happens in the second step of PCR?

Annealing

• Cooling to 54°C for 30-60 seconds allows for the specific primers to bind at targeted locations

• Excess primers are added to prevent single strands from pairing up again

What happens in the third stage of PCR?

Elongation

• Heated to 72°C which is optimum temperature for taq DNA polymerase to add new nucleotides and make new strands of DNA

What’s gel electrophoresis?

Technique used to separate fragments of DNA (or proteins) according to size and charge

An electrical current is used to attract charged fragments through a gel

How does gel electrophoresis work?

1) Special gel is set up with wells cut out at one end, samples of amplified DNA fragments are placed in wells

2) The shallow tank is filled with an electrolyte with electrodes at either end, voltage applies created an electrical field

3) The negatively charged DNA fragments are attracted to the positive anode and move towards that end. The gel restricts movement so speed with which the fragments travel is dependent on size

4) After set amount of time, current is stopped and gel is removed. A special dye is used to make the DNA visible.

5) The bands that can be observed show that fragments of the same length travel the same distance and would appear as a match

Which DNA fragments travel far in gel electrophoresis and why?

• Smaller DNA fragments bc are attracted to anode and move through the gel easier bc are lighter and less surface area

Applications of PCR and gel electrophoresis

• Compare DNA of different individuals in forensic investigations and cases of disputed paternity

Extensive steps to apply PCR and gel electrophoresis to paternity or forensics

1) Obtain DNA, e.g. DNA from evidence such as sample of blood, sexual fluids or hair follicles, or DNA from suspects like cheek swabs

2) Amplify and select particular regions of DNA using PCR, need to amplify the sections of DNA for meaningful and reliable analysis, need to select the varying short tandem repeats for high degree of confidence when distinguishing

3) Gel electrophoresis to seperate DNA fragments to compare

4) Analysing the profile, comparing the pattern of bands between suspects and from crime scene or between a child’s profile and potential parents