D1.1 DNA Replication Flashcards
What is DNA replication?
The production of exact copies of DNA with identical base sequences
Outline the purposes of DNA replication
Growth: involves the addition of new cells to make an organism larger, each new cell requiring a complete copy of the organisms DNA through DNA replication that must occur prior to cell division.
Reproduction
Repair: new cells need to be produced to replace the damaged/destroyed ones, DNA replication occurs before cell division. As they need to be exactly like the ones around them, they also need to be differentiated.
Describe what is meant by semi-conservative nature of DNA replication
1) As the double strands of DNA come apart, each strand acts as a template for DNA replication
2) The new stands are synthesised by adding nucleotides one by one and linking them together
3) When replication is complete each new double strand synthesised will contain an original stand of DNA
What is the role of complementary base pairing in DNA replication?
To maintain and conserve the correct sequence of bases in the molecule, ensuring newly synthesised strands are exact copies of the original (ensures genetic code remans intact between generations).
Complementary bases form H-bonds which stabilise the structure.
(A-T, C-G)
What experiment proved that DNA replication is semi-conservative?
Meselson and Stahl differentiated between different DNA theories by using a density-labelling method to track DNA replication
They found that each newly replicated DNA molecule consisted of one old and one new strand, thus confirming the semi-conservative model of DNA replication.
Why must DNA strands be separated prior to replication?
The two strands of the parent DNA molecule must separate so they can each serve as a template for the new DNA strands being built.
What is the role of helicase in DNA replication?
The enzyme helicase unwinds the DNA double helix and separates the two strands by breaking H-bonds between the bases.
ATP is used to expose these bases that are usually protected within the molecule.
What is the role of DNA polymerases in DNA replication?
Once strands are separated and bases exposed, the enzyme DNA polymerase moves along the separate DNA strands (towards the replication fork), using them as templates.
Then, it will begin building the new strand of DNA by placing and attaching fee nucleotides in a chain.
[Overall is responsible for synthesis of DNA molecules during DNA replication]
Are there seperate DNA polymerases for each strand of template DNA?
Yes
What is the polymerase chain reaction (PCR)?
A technique used to amplify and separate small fragments of DNA and copy these specific sections in a cell sample.
Outline the 3 step process of the PCR
In a thermal cycler temperature variations are used to control DNA replication:
- Denaturation— temperature is increased (98degreesC) to separate the DNA strands
- Annealing—temperature is decreased (55degreesC) allowing RNA primers (short RNA sequences) to serve as starting points for DNA polymerase, attaching to the 3’ ends of the target sequence
- Elongation— a heat resistant DNA polymerase (Taq) binds to the primer and copies the strand by adding free nucleotides (72degreesC/2mins)
What is Taq polymerase and why is it so important?
A heat-resistant DNA polymerase frequently used in the PCR.
It is used because the enzyme can withstand high temperatures without denaturing that are required in the PCR.
Outline the function of the 4 components needed to carry out the PCR: Primers, Taq polymerase, Nucleotides, Buffer/cofactors
DNA template—
Primers— short RNA sequences that serve as a starting point for DNA syntheses by Taq
Taq polymerase— heat-resistant enzyme that synthesises DNA
Nucleotides— many and free, used by Taq to synthesise new DNA strands
Buffer/cofactors—maintain pH balance of the mixture for optimal enzyme activity
Deduce the number and relative size of DNA fragments from the number of bands in an electrophoresis gel
What is gel electrophoresis?
A technique used to separate and analyse DNA fragments based on their size and charge using an electric field and gel matrix.
Outline the 3-step process of DNA electrophoresis
[Before GE is carried out a DNA sample must be amplified using PCR]
- DNA is then cut using restriction enzymes, which act like molecular scissors, cutting the DNA at specific points
- DNA samples are placed in a well on a gel acting as a molecular sieve/filter (gel consists of mesh filaments)
- The sugar phosphate backbone of DNA is negatively charged: once the gel is immersed in a connecting fluid, electricity is applied and DNA samples move towards positive electrode
- The movement of the samples and distance is restricted by molecular size of DNA —> the smaller the fragments, the furthest down the gel
How and why do DNA fragments separate during electrophoresis?
The gel is porous, and the DNA must travel through the spaces within the gel.
Smaller pieces can slip through the spaces more easily, allowing them to travel further along the gel in a given amount of time.
Usually, one or more of the wells is filled with a ‘DNA ladder’, which contains DNA fragments with a range of known lengths.
By using the DNA ladder, the length of sample fragments can be determined.
List the applications of PCR and electrophoresis
Viral infection testing— eg. Covid testing
DNA profiling—a technique that examines variable portions of DNA to create a profile or ‘fingerprint’ that is unique to the individual.
[Every cells contains our entire genome, and we shed cells continually, leaving them in the environment around us]
What are the advantages (3) and disadvantages (3) of the use of PCR to test for viral infection?
Advantages— highly specific, high sensitivity, rapid detection
Disadvantages—high cost, technical complexity, does not differentiate between active and inactive infections
Outline the process of DNA profiling
Most genomes have short, repeated DNA sequences called tandem repeats (they do not code for proteins: STRs&VNRs).
Number of repeats vary between individuals in a population, allowing for a specific fingerprint for every individual.
What are VNTRs and STTRs?
Sections of DNA that are repeated a number of times (AGGAGGAGGAGG)
Outline the process of DNA profiling with VNTRs
Each individual inherits 2 alleles from every gene (or VNTR) they receive from both parents, this allows for no one having the same VNTR combination.
The combination of alleles is the basis of DNA profiling as VNTR shows variations between individuals in terms of the number of times the sequence is repeated.
Outline the process of DNA profiling with STRs
STRs are specific sites in the DNA where highly variable numbers of a short, repeated sequence are found.
An individual inherits one copy of STR from each parent, giving either a heterozygous or homozygous genotype, depending on whether there are similar repeat sizes.
Typically used and compared to create the genetic fingerprint of an individual.
What are the 5 steps for making a DNA profile?
- DNA sample is collected (blood, semen, saliva), extracted and amplified using PCR
- Satellite DNA are cut with a specific restriction enzymes to generate fragments
- DNA fragments are separated using gel electrophoresis
- Fragments length will differ between individuals due to the variable length of STRs
- A pattern of bands of DNA is produced on the gel— this is the individuals DNA profile and can be compared with other samples using a DNA ladder