Unit 2 - Nucleic acids & DNA replication Flashcards
What does DNA stand for?
Deoxyribonucleic acid
What does RNA stand for?
ribonucleic acid
What is the monomeric unit for nucleic acids?
Nucleotides
What are the 3 components of a nucleotide?
- phosphate group
- pentose (5 carbon sugar) either deoxyribose or ribose
- nitrogenous base
Name the 5 nitrogenous bases
- thymine
- guanine
- adenine
- cytosine
- uracil
Name the 4 bases present in DNA
- thymine
- adenine
- guanine
- cytosine
Name the 4 bases present in RNA
- uracil
- adenine
- guanine
- cytosine
Which base is never found in DNA?
Uracil
Which base is never found in RNA?
Thymine
How are polynucleotides formed?
- condensation reactions occur
- between the phosphate on carbon 5 of one nucleotide and the hydroxyl group on carbon 3 of another nucleotide
- water is released
- phosphodiester bond formed
What sort of bond is formed between nucleotides?
Phosphodiester
How are sequences of RNA/DNA given?
From the 5’ end to 3’ end
What are the two types of bases?
Purines and pyrimidines
What is the structure of purines?
Double ringed
What is the structure of pyrimidines?
Single ringed
Which bases are purines?
Adenine and guanine
Which bases are pyrimidines?
Cytosine, thymine, and uracil
What does complementary base pairing allow?
-DNA to be copied and transcribed
What type of bonds hold bases together?
Hydrogen bonds
How many hydrogen bonds form between Adenine and Uracil/Thymine?
2
How many hydrogen bonds form between Guanine and Cytosine?
3
In order to allow for the bases to form hydrogen bonds what must happen to one of the polynucleotide chains?
It must be rotated through 180 degrees
How does the sugar phosphate backbone run on one of the polynucleotide chains? How does the other polynucleotide chain run? What is this called?
From 5’ to 3’ and the other from 3’ to 5’
The antiparallel nature of DNA
Describe the steps involved in extracting DNA
- sample ground up using pestle and mortar
- sample mixed with detergent
- salt is added
- protease is added
- ice cold ethanol is added down the side of the test tube
Why is the sample ground in the extraction of DNA?
it breaks down the cell walls
Why is detergent added to the sample in the extraction of DNA?
breaks down the cell membrane, releasing the cell contents into solution
Why is salt added to the sample in the extraction of DNA?
neutralises charges on phosphates in sugar phosphate backbone
breaks the hydrogen bonds between DNA and water
makes DNA less soluble
Why is protease added to the sample in the extraction of DNA?
breaks down the proteins associated with DNA
Why is ethanol added to the sample in the extraction of DNA?
Causes the DNA to form a white precipitate between the layer of the sample and the ethanol
Why is a low temperature maintained throughout the process of DNA extraction?
Reduces rate of enzyme controlled reactions that break down DNA
Why should the ethanol be ice cold?
Helps to make DNA more insoluble
At what point in the cell cycle does DNA replication occur?
S phase
Why is it important that DNA replication results in 2 genetically identical molecules of DNA?
- ensures the continuation of species
- ensures cells and structures in a species are maintained
Why is DNA replication described as being semi-conservative?
- Each one of the two daughter molecules contains one original strand of DNA and one newly synthesised strand
- the original strand acts as a template for a new strand
What is the relationship between the two daughter molecules?
They are genetically identical
What is the relationship between the daughter molecules and the original DNA molecule?
they are identical
Which two enzymes are used in DNA replication?
DNA helicase
DNA polymerase
What is the role of DNA helicase?
- unwinds DNA from histones
- unzips DNA by breaking Hydrogen bonds betwem complementary base pairs
What is the role of DNA polymerase?
-catalyses the reaction to form phosphodiester bonds between the free nucleotides to form a sugar phosphate backbones
Outline how DNA is replicated
- DNA helicase unwinds DNA from histones and unzips DNA by breaking Hydrogen bonds between complementary base pairs
- free nucleotides are attracted to the exposed nucleotides on the strand of DNA by base complementarity
- DNA polymerase forms phosphodiester bonds between nucleotides
- two genetically identical molecules of DNA are formed
What does the unzipping mean for DNA nucleotides?
They are exposed
Due to the nucleotides being exposed, what happens?
Free nucleotides are attracted to the exposed nucleotides by base complementarity and form hydrogen bonds
Where do the free nucleotides come from?
Exogenous come from our diet.
We can also synthesis nucleotides.
How do free nucleotides enter the nucleus?
From the cytoplasm through the nuclear pores
What is a primer?
A short piece of RNA or DNA with a complementary sequence to part of the DNA template
What direction does DNA synthesis occur?
5’ to 3’
What is the difference between the leading strand and lagging strand?
The leading strand is built continuously whereas the lagging strand is built discontinuously
Why is there a leading strand and a lagging strand?
The antiparallel nature of DNA
Why are primers needed in DNA replication?
DNA polymerase requires them in order to bond to the 3’ OH group
What does the discontinuous building of the lagging strand lead to the production of?
Okazaki fragments
Which experiment was used to prove that DNA replication is semi-conservative?
The Meselsohn and Stahl experiment
Which two techniques did the Meselsohn and Stahl experiment use?
Mass labelling and density centrifugation
Which isotope was used for mass labelling in the Meselsohn and Stahl experiment?
N- 15
Why was N-15 used in the Meselsohn and Stahl experiment?
It could be incorporated into the nitrogenous bases and therefore track the movement of them
Which part of the nucleotide was labelled in mass labelling?
The nitrogenous bases
How did density centrifugation work in the Mehselson and Stahl experiment?
- A CsCl gradient is produced by centrifugation
- more dense molecules move to the bottom and lighter molecules to the top
- When DNA is placed into this solution it migrates to the position in the gradient with the same buoyant density
DNA was placed in a solution of what in the Meselsohn and Stahl experiment?
CsCl (caesium chloride)
Outline the two control experiments in the Meselsohn and Stahl experiment
- E.coli placed in growth factor containing N-15 (heavy nitrogen) which the E.coli incorporated into their DNA, DNA extracted and centrifuged with CsCl
- E.coli placed in growth factor containing N-14 (normal nitrogen) which the E.coli incorporated into their DNA, DNA extracted and centrifuged with CsCl
What was seen in the two control experiments in the Meselsohn and Stahl experiment?
N-15, the DNA settled further down the density gradient whilst the N-14 DNA was higher up the density gradient
Why was E.coli used in the Meselsohn and Stahl experiment?
- the environment in which it grows can be carefully controlled
- it would incorporate the different isotopes of Nitrogen into its DNA
- it divides every 20 minutes
Why were the control experiments important to the Meselsohn and Stahl experiment?
- They allowed the position of where each N isotope sits after centrifugation depending on its density to be identified
- if there was a mixing of the two isotopes a band would be seen inbetween
What was the key experiment in the Meselsohn and Stahl experiments?
- E.coli was grown in a growth media containing N-15 for several divisions
- it was then transferred to a growth media containing N-14 and only allowed to divide once
- the DNA was then extracted and centrifuged with CsCl
What was observed in the key Meselsohn and Stahl experiment?
A band of DNA was formed inbetween the N-15 and N-14 bands
How did the key Meselsohn and Stahl experiment prove DNA replication is semi-conservative?
- the initial molecule of DNA contained only N-15 nucleotides
- as DNA replication is semi-conservative the DNA which was newly synthesised in the N-14 growth media would contain one strand with N-15 nucleotides and one strand with N-14 nucleotides
- so the band would be in the middle
If DNA replication was conservative what would have been observed in the key Meselsohn and Stahl experiment?
A band of DNA at the N-14 and N-15 positions
If the E.coli were allowed to divide twice in the key Meselsohn and Stahl experiment in N-14 what would be seen?
A band at N-14 and in the middle in the ratio 1:1
If the E.coli were allowed to divide three times in the key Meselsohn and Stahl experiment in N-14 what would be seen?
A band at N-14 and in the middle in the ratio 3:1
Explain why complementary base pairing is important in DNA replication
- reduces occurrences of mutation
- allows for formation of hydrogen bonds
- DNA is replicated without error
Suggest 3 precautions that Meselson and Stahl would have taken in order to ensure that the centrifugation part of their investigation produced valid results
- the tubes were spun for the same amount of time
- the tubes were spun at the same speed
- the solution the DNA is put in is the same concentration
What is a mutation?
A change in the sequence of DNA bases within a gene
How do mutations occur?
A base changing
What are 2 possible consequences of mutations?
- evolution
- genetic disorders
Give 3 differences between DNA and RNA?
-DNA has a double stranded structure whilst RNA has a
single stranded structure
-In DNA A bonds to T and G bonds to C, in RNA A bonds to U and G bonds to C
-DNA is too big to leave the nucleus, RNA is small enough to leave the nucleus