D1 Flashcards
What is DNA replication?
The production of new DNA strands using existing strands. Involves seperating strands and then using the single strand as a template for a new DNA strand
What is meant by “semi-conservative” in reference to DNA replication?
It refers to the fact that at the end of each round of DNA replication, each double helix contains one “old” strand of DNA, and one new strand from replication
What are the two processes requiring DNA replication
- Growth and repair of tissue- DNA replication before mitosis
- Reproduction- DNA replication before meiosis produces sex cells
How does complimentary base pairing facilitate DNA replication?
Specific base pairing rules mean that each original strand can serve as a template for its complimentary strand. This allows semi-conservative replication to occur.
How did the Messelsohn- Stahl experiment show that DNA replication is semi-conservative?
They used nitrogen isotopes to track original DNA through generations to show that replication is semi-conservative. Original was marked with N15 and new marked with N14. Demonstrated that N15 always remained but decreased over time, therefore is semi-conservative
Explain the role of helicase in DNA replication
Helicase is a ring-shaped enzyme that pulls one strand of DNA through its ring to break the hydrogen bonds between base pairs. This seperates the original DNA strands.
Explain the role of DNA polymerase III in prokaryotic DNA replication
DNA polymerase is a group of enzymes that work off the primers to attach new DNA base using free nucleotides in the nucleus, and pairing it with the template strand using base pairing rules to build 5’ to 3’ strands on the leading and lagging strands. This then hydrogen bonds between complimentary bases to build the new strand which is also proofread to check for mistakes and mutations.
What is the conservative theory of DNA replication? (disproven)
Original strand stays together, so in each generation there will be one full original double helix
What is the dispersive theory of DNA replication? (disproven)
Original DNA is spread randomly throughout the subsequent generations.
What is a replication fork?
The place where DNA is currently being seperated into 2 single strands. It is where helicase is. There is 2 replication forks heading in each direction of the origin (where it started being split)
What is a primer?
RNA primer is placed to indicate where DNA polymerase III must begin the new strand of DNA. In the leading strand, only one primer is placed, but in the lagging strand, multiple primers must be placed.
What is the leading strand in DNA replication?
The leading strand is the original DNA strand that runs 3’ to 5’, so that the new strand can easily be built 5’ to 3’, as new bases can only be added to the 3’ end of the new strand. It can be built continuously, therefore is faster.
What is the lagging strand in DNA replication?
The lagging strand is the original strand that runs 5’ to 3’. As new bases can only be added to the 3’ end of the new strand, the lagging strand must be built backwards in fragments which are combined later. This means it is slower, and thus lags behind.
What are Okazaki fragments?
Okazaki fragments are short, copied fragments resulting from building lagging strand backwards and in sections. These are glued together later to create a complete strand.
What is DNA proofreading?
DNA proofreading ensures correct placement of complimentary bases and reduces mutations. In prokaryotes, this is done by DNA polymerase III which cuts out mistaken base pairs, through exonuclease activity.
What is the difference between the 5’ and 3’ end of a nucleotide?
3’ refers to the 3rd carbon of the pentose sugar on the DNA nucleotide, while 5’ refers to the 5th carbon, where the P group is attached.
What are the limitations of DNA polymerases when building new strands of DNA?
- It cannot initiate a brand new strand of DNA- can only add to an existing nucleotide, thus needs primers to build new strand
- It can only add a new nucleotide to the 3’ end of existing nucleotides, thus can only build 5’ to 3’.
How does the directionality of DNA polymerase lead to a leading and lagging strand in replication?
DNA polymerase III can only add bases to the 3’ end, so it cannot use the first primer on the 5’ to 3’ strand, thus a second primer must be placed, and the fragment can be built from the second primer to the first primer.
What is the role of DNA primase in prokaryote DNA replication?
DNA primase builds and places the correct complimentary RNA primer along the original DNA strand to initiate replication. For leading strands, only one primer is required, but in lagging strands, multiple primers are needed.
What is the role of DNA polymerase I in prokaryotic DNA replication?
DNA polymerase I removes RNA primers in the lagging strand, acting as an exonuclease and then adding new DNA nucleotides, acting as a polymerase.
Note: it cannot seal the gaps between the Okazaki fragments and the primers.
What is the role of DNA ligase in prokaryote DNA replication?
DNA ligase repeats the lagging strand by creating phosphodiester bonds between Okazaki fragments so they appear as a continuous DNA strand.
Explain the steps of DNA replication
- Helicase seperates the strands of DNA by breaking hydrogen bonds
- Gyrase prevents supercoiling of the helix ahead of helicase
- Single Stranded Binding Proteins (SSB’s) hold newly seperated strands apart
- DNA primase builds and places complimentary RNA primers at the origin, and along the lagging strand
- DNA polymerase III adds complimentary DNA bases to a free 3’ end of a nucleotide to build leading strand and Okazaki fragments, and proofreads new strand
- DNA polymerase I removes RNA primers (exonuclease) and replaces with DNA primers (polymerase)
- DNA ligase creates phosphodiester bonds between Okazarki fragments
What is a thermocycler?
A thermocycler is a small machine that runs multiple PCR samples simeltaneously by using quick heating and cooling metal wells to PCR can rapidly generate DNA.
What are primers in PCR?
Primers in PCR are made of DNA and are used to identify the section of DNA to be copied and start the new strands for polymerase to act on.
What is Taq polymerase?
Taq polymerase is the polymerase used in PCR. It is adapted to hot environments so it functions optimally at 72 degrees, the temp in the final stage of PCR.
What is Gel Electrophoresis?
Gel electrophoresis allows negative DNA fragments to move through agarose gel to a positive electrode, which is stopped before all reach the end. Smaller fragments move faster than bigger ones so the fragments are scattered by size.
What is DNA profiling?
DNA profiling is the process of creating a unique DNA banding pattern using restriction enzymes to cut DNA, then gel electrophoresis seperates the fragments by size. It can be used in forensics, paternity testing and genetic screening
What are short tandem repeats?
STR’s are short 2-7 base pair fragments that are repeated, but the number of repeats varies widely between individuals.
What is the purpose of PCR?
To take small amounts of DNA and create many copies often of a specific fragment. PCR allows a small amount of DNA to generate and produce visible strands on gen after electrophoresis
Explain the steps of PCR, including what happens and the temperatures.
- Denaturation to seperate the double stranded DNA into 2 strands by heating to 95 degrees to break H bonds b/w bases
- Annealing- drop temperature to 54 degrees and lots of primers are added to bind to the target sequences to show taq polymerase where to start copying
- Extension- heated to 72 degrees which is taq polymerase’s optimum temperature. Taq polymerase adds complimentary free nucleotides next to the primer to build new complimentary strands
How does electrophoresis seperate fragments by size?
DNA has a negative charge, so it is attracted to the positive electrode. It is placed in agarose gel, which is porous so the DNA can move through. Smaller fragments move fast, larger fragments move slower. Thus they become seperated by size
How is PCR used to test for Coronavirus?
Viral DNA is turned into DNA by reverse transcriptase, primers used to copy fragments only in the virus. Markers bind to viral DNA if present, once PCR makes enough fragments
How is PCR and electrophoresis used for paternity testing?
DNA samples from mother, child and potential fathers are taken. PCR replicates short tandem repeats which are then used in gel electrophoresis. The child should have the same number of repeats as the mum or dad. Bands the child has but the mother doesn’t have must come from the father.
What is gene expression?
Gene expression is the using of the instructions in a gene. Synonymous with protein synthesis.
What is a gene?
Agene is a segment of DNA that codes for one protein