DNA replication, D1.1

• What is DNA replication?

  DNA replication is the production of new DNA with base sequences identical to existing strands.

  Takes place during the S phase of interphase

  DNA helix is unwound and unzipped by helicase, which breaks the hydrogen bonds between base pairs

  The exposed, unpaired bases form a template

  Free nucleotides are added by complimentary base pairing to the exposed bases by DNA polymerase

  ATP is needed for this, as a source of energy

  Two identical DNA molecules are produced as a result Because each new DNA molecule retains half of an old one, this is known as the 'semi-conservative hypothesis
• What is a replica, and how does this relate to DNA replication?

  A replica is an exact copy of something

  DNA replication is the production of new DNA with base sequences identical to existing strands.

  DNA has been replicated since LUCA
• Why is DNA replication necessary?

  DNA replication is needed for two biological processes:

  
  

  Reproduction and growth. Reproduction:

  Offspring need copies of the base sequences of their parents

  Parents must replicate DNA to reproduce

  
  

  Growth and tissue replacement in multi-cellular organisms:

  Each cell needs a full set of the organism's base sequences

  So before a cell can divide, it must replicate all of its DNA

  Cell division is needed to replace cells in tissues
• What is semi-conservative replication?

  During DNA replication, the DNA strand is unzipped by DNA helicase, forming a template. New nucleotides are added via complimentary base pairs to the template, which is the polymerisation of the new DNA strand. The site where the copying is actively occurring is known as the replication fork. After DNA replication, there are two DNA molecules, both composed of an original strand and a newly synthesised strand. Therefore, DNA replication is semi-conservative, as each new DNA molecules partially consists of (conserves) an old one.
• What is the role of complimentary base pairing in DNA replication?

  Some of the nitrogenous bases fit together perfectly, making a complete whole. In DNA, Adenine and Thymine pair together, linked via two hydrogen bonds. Cytosine and Guanine pair together, linked via three hydrogen bonds. Complimentary base pairing ensures that the two DNA molecules resulting from DNA replication are identical in their base sequences to the parent molecule. It also ensures a high degree of accuracy when new strands are assembled on a template strand. It ensures that only 1 in 10 billion bases is incorrect when DNA is replicated. A diploid cell has approximately 6 billion base pairs, so there are on average only 0.6 errors when all the DNA is replicated . This explains the genetic continuity that exists between generations.

  Accuracy

  Template function

  Replication efficiency
• What is the replisome?

  DNA replication is a multi-stage process carried out by an assemblage of functional subunits known as a replisome. Helicase and DNA polymerase are two proteins that are essential parts of replisomes.
• What is helicase?


  Ring-shaped protein

  Separates the two strands of DNA so that they can act as templates for the new strand. Without separation, the base sequences would not be accessible for copying.

  Breaks hydrogen bonds between bases, allowing one strand to be pulled through the hole in helicase's ring and the other to pass to the side of it. This creates the replication fork.

  Uncoils the double helix

  Unwinding/unzipping of the double helix DNA The unwinding of DNA causes tensions that could cause supercoiling. To prevent this, the tensions are relieved by parts of the replisome.
• What is DNA polymerase?

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

  Separate DNA polymerases are used for each strand, all which are within the replisome. One polymerase synthesizes the leading strand continuously, while another synthesizes the lagging strand discontinuously in short fragments (Okazaki fragments).

  Add one new nucleotide at a time

  When DNA polymerase brings a new nucleotide to the template strand, a hydrogen bond may not always form, if the base is not complimentary. If this is the case, the nucleotide will break away and a new nucleotide will be brought into position.

  Links the nucleotide at the end of the new strand to the template strand to form the double helix- a covalent bond is formed between the phosphate group of the new nucleotide and the deoxyribose of the template strand's nucleotide.
• What is PCR?


  The polymerase chain reaction is also known as PCR, and is an automated method of DNA replication. This means that it is operated by a PCR machine. Its function is the amplify DNA (producing more DNA from a specific base sequence). A small amount of DNA is inputted, and the PCR cycle cycles through steps that are triggered by temperature changes to output a large amount of DNA replicas. The quantity of DNA is doubled each cycle.

  The PCR cycle may also be known as a thermocycler.

  The optimal length of a DNA sequence for amplification is 100 base pairs.
• What does a typical PCR consist of?

  1- Melting

  Heating to 95 degrees celsius breaks the hydrogen bonds that hold the DNA strands together, but does not break the covalent bonds between the nucleotide components. This separates it into two single strands.

  2- Annealing

  Cooling to 54 degrees celsius allows primers to bind, enabling rapid binding and preventing the single strands of DNA from pairing up again.

  3- Elongation

  Heating to 72 degrees celsius provides optimum temperature for Taq DNA polymerase, which binds to the single-stranded DNA adjacent to the primer. It assembles the new DNA strands.
• What is a primer?

  A primer is a short single strand of DNA (or RNA) that provides a starting point for DNA synthesis.

  In PCR, primers are designed to match the beginning and end of the target DNA sequence so that the DNA polymerase knows where to start copying.

  Therefore only selected sequences of DNA may be replicated.
• What is Taq DNA polymerase?


  Taq DNA polymerase is a special enzyme used to build new DNA strands during PCR (Polymerase Chain Reaction).

  It comes from a heat-resistant bacterium called Thermus aquaticus, which lives in hot springs. Because of this, Taq polymerase can work at high temperatures (like 72°C), which is important for PCR since the DNA is repeatedly heated and cooled.

  Its main job is to add new DNA bases to a growing strand, using the original strand as a template.
• What is Gel electrophoresis?

  Gel electrophoresis separates DNA molecules by length.

  A sheet of gel with gaps (wells on one end is placed in a shallow tank with electrodes at both ends. The wells are next to the negative cathode.

  The gel is covered with an electrolyte solution.

  The DNA sample is placed in the well.

  A voltage is applied across the electrodes to create an electric field. This makes charged molecules move through the gel. As DNA has a negative charge (due to the phosphates) it moves towards the positive anode.

  The gel resists the movement of molecules, allowing small molecules to move faster than larger ones.

  When DNA moves from its well close to the cathode to the anode, it separates by length, with longer chains moving slower than shorter chains.

  When the smallest DNA molecules have barely reached to anode, voltage is switched off and the gel is removed.

  The tank is treated with a dye that makes the DNA visible.

  The DNA moves in a series of parallel lanes, the number of bands in each lane indicating how many different lengths of DNA were in the sample, as each band is a group of DNA molecules of the same length. A ladder (DNA fragments of known lengths) may be placed near the lane, to estimate the lengths of the DNA molecules within the bands.
• What do the results of gel electrophoresis indicate?

  Number of bands = Number of different DNA fragments present.

  Relative size of fragments = Inversely related to how far they travel in the gel.

  Smaller fragments move further and faster through the gel.

  Larger fragments move more slowly and stay closer to the wells.
• How does coronavirus testing work?

  Swab taken from nose or throat

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

  RNA in the liquid sample is converted to DNA using the enzyme reverse transcriptase

  PCR amplifies specific viral base sequences that are markers of the strain of coronavirus. Around 35 PCR cycles occur.

  Fluorescent markers are attached to the DNA produced, and if the levels of fluorescence rises above a target level, the result of the test is positive.
• What are the advantages of coronavirus testing?

  Very sensitive, as one molecule of RNA is amplified to produce around 35 billion DNA molecules

  Very specific, as primers can only detect one strain of the virus
• What are the disadvantages of coronavirus testing?

  Requires relatively expensive materials and equipment only available in testing labs

  Results may not be readily available as thermal cycling takes some time
• What are short tandem repeats?

  DNA profiling distinguishes between individuals by using base sequences known as short tandem repeats (STRs).

  These are short sequences of DNA, usually between 2 and 7 bases long, that are repeated one after another in a row.

  Each person has these repeats in many places across their chromosomes, but the number of repeats is different from person to person.

  For example:

  On chromosome 5, the sequence TAGA might be repeated 6 to 15 times in a row.

  On chromosome 18, the sequence AGAA might be repeated 7 to 27 times.

  Even though many people have the same STR sequences, the number of repeats at each location is very individual, like a genetic fingerprint.
• How does DNA profiling work?

  A sample of DNA is obtained

  Selected short tandem repeats in the sample are copied by PCR (at least 13)

  The DNA produced by PCR is separated according to length of fragments and therefore numbers of repeats using gel electrophoresis

  The appearance of bands of DNA on the gel is always the same for all DNA samples taken from the same individual, and is known as one's unique DNA profile.