DNA replication
the production of exact copies of DNA with identical base sequences.
explain the semi-conservative nature of DNA replication
Each new double strand of DNA produced contains one strand of the original DNA (template strand) and one strand of newly synthesized DNA.
This occurs because each stand of the original DNA acts as a template for the new strand to be built from.
Importance of complementary base pairing in DNA replication
The complementary base pairing rule ensures that the new strands that are built are exact copies of the of the original.
Role of helicase
Unwinds the double helix and separates/unzips the two DNA strands by breaking the hydrogen bonds between the bases. The separation of the strands exposes the bases usually protected within the molecule. Single strand binding proteins prevent the reformation of hydrogen bonds.
Role of DNA polymerase III
DNA polymerase (not limited to III) is responsible for synthesizing DNA molecules during DNA replication.
Moves along the separate DNA strands, using them as templates.
Assembles the new strands of DNA by placing free nucleotides in the correct sequence in a chain according to the base sequence of the template strand and the complementary base pairing rule. (the new strands are assembled by DNA polymerase III in a 5’ to 3’ direction.)
DNA polymerase III also proofreads.
Role of gyrase
Relieves tension and supercoiling generated ahead of the replication fork (gyrase moves ahead of helicase)
Role and function of DNA primase (type of RNA polymerase)
attaches small RNA primers made of several RNA nucleotides to the template strand. The primer allows DNA polymerase III to attach and begin assembling the free nucleotides into the new strand of DNA.
Only a single primer is required on the leading strand as it is continuous.
On the lagging strand, primers need to be placed at regular intervals to attach polymerase III at multiple points (discontinuous).
DNA polymerase I
Removes the RNA nucleotides of the primers and replaces them with the correct DNA nucleotides.
DNA ligase
Catalyses the formation of the phosphodiester bonds between the Okazaki fragments, making the replicated strand —-
DNA proofreading - when, which enzyme, how
As a new DNA strand is being formed, DNA polymerase III proofreads.
If a nucleotide is placed with a mismatched base, the incorrect nucleotide is removed and replaced with the correct nucleotide. The nucleotide is removed from the 3’ terminal.
Polymerase chain reaction (PCR)
Laboratory technique used to amplify a specific DNA sequence/small fragments of DNA, producing multiple copies of it.
The discovery of what technique has revolutionalised medical and forensic science and molecular biology?
PCR
What does the PCR reaction chamber contain
many free nucleoside triphosphates
primers
Taq polymerase (a short DNA/RNA sequence) (Taq stands for Thermophyllus aquatic, which lives in hot springs)
The desired DNA section is placed in the reaction chamber
process of PCR
- denaturation phase
DNA is heated enough to break the hydrogen bonds that hold the two strands of the DNA double helix together (around 98 degrees Celsius) - annealing phase
As temperature is cooled to around 60 degrees, primers bond to complementary sequences in the DNA sample. - extension phase
Taq polymerase replicates DNA using the primer as a starting point to extend from (around 72 degrees)
^one cycle complete.
Once the replication has been completedd, the strands are heated to separation and the process begins again.
The amount of DNA doubles each time a cycle occurs, resulting in exponential growth. Within a few hours, billions of copies of the DNA sequence would be created.
Gel electrophoresis
A technique that can be used to identify some key features of the DNA
How does gel electrophoresis work (1)
gel electrophoresis uses an electrical current to move molecules through a semisolid medium or (porous) gel. The DNA molecules are separated by size and amount of charge.
DNA molecule charge
negative
Gel electrophoresis process
- Restriction enzymes (restriction endonucleases) cut the backbone of the DNA double helix at the restriction sites, producing shorter DNA segments and distinctive fragment patterns. Since each individual has different DNA the location of these restriction sites vary, meaning different individuals end up with different sized fragments when cut.
- the DNA samples are placed in wells/small depressions on the negative end of the gel. The gel is located in a buffer filled chamber that allows electrical charges to move.
- When the chamber is turned on, an electrical current runs through the gel, and DNA moves towards the positive electrode. DNA fragments don’t move at the same speed.
- The end result is a banding pattern. If one of the wells is filled with a DNA ladder, the length of sample fragments can be determined from the DNA ladder. two DNA samples of the same individual will also have the same banding pattern.
A dye is used in the gel that binds to DNA since DNA fragments don’t have colour.
What are restriction sites
highly specific sequences on the DNA double helix where restriction endonucleases bind and cut.
what differentiates the speed that the DNA fragments move in gel electrophoresis
Size/length of the DNA fragments.
Smaller fragments can move faster because they can easily move through the pores in the gel, while bigger fragments move slower.
What is a DNA ladder
A solution of DNA molecules which contains DNA fragments of known lengths. The length of sample fragments can be determined from the DNA ladder by comparing it against the ladder banding pattern (acts as a reference).
What is the purpose of PCR
To provide enough copies for gel electrophoresis and/or other tests.
Application of PCR and gel electrophoresis
Testing for and diagnosing inherited diseases
DNA profiling (paternity testing, forensic investigations, etc.)
Studying biodiversity
Determining which strains of bacteria causes more serious illness
etc.
How can PCR and gel electrophoresis be used for DNA profiling
DNA profiling examines variable portions of DNA to create a profile/’fingerprint’ unqiue to the individual.
Most genomes have tandem repeats, and the number of repeats can vary greatly between individuals. Restriction enzymes are used to chop the DNA into fragments that vary in length depending on the repeats.
After amplifying with PCR, the mix of DNA fragments is separated using gel electrophoresis.
There must be the same number and length of DNA fragments to match the DNA from evidence to individual.
It is possible that there are other individuals with the same combination of alleles, so several dozen sites may be analysed.
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