Lecture 7- DNA and RNA Flashcards
(47 cards)
1
Q
What are the 3 components of nucleotides
A
- A nitrogenous base
- A 5-carbon sugar - ribose or deoxyribose
- At least 1 phosphate group
2
Q
What are the different nitrogenous bases
A
- Adenine
- Guanine
- Cytosine
- Thymine
- Purines- 2 Rings - A, G
- Pyrimidine - 1 ring - T, C
3
Q
Define a nucleoside
A
- A nitrogenous base covalently linked to a 5-carbon sugar
4
Q
Define a nucleotide
A
- A nucleoside plus a phosphate group
- Is a constituent of DNA and is called deoxyadenosine monophosphate
5
Q
Describe role of nucleotide in metabolism
A
- Nucleotide carry packets of chemical energy in the form of nucleoside triphosphates
- Adenosine triphosphate (ATP)
- Guanine triphosphate (GTP) etc
- When ATP is consumed in a metabolic processes it converts to either the di or mono phosphates; ADP and AMP respectively
6
Q
Describe discovery of DNA
A
- First isolated by Miescher in 1869
- Molecular structure of DNA was first identified by Watson and Crick in 1953, guided by X-ray diffraction data obtained by Franklin
7
Q
What are the two types of DNA in the human body
A
- Nuclear
- Mitochondrial
8
Q
Describe the structure of DNA
A
- DNA is a polynucleotide
- Consists of two biopolymer strands coiled around each other to form a double helix
- The two strands of DNA run in opposite directions to each other and therefore anti-parallel
- Both strands of DNA store the same biological information as are identical
- Within eukaryotic cells DNA is organised into chromosomes
9
Q
How are nucleotides joined together in DNA
A
- Joined in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next
- Results in an alternating sugar-phosphate backbone
10
Q
Describe DNA base-pairing
A
- The nitrogenous bases of the two separate polynucleotide strands are bound together according to base-pairing rules with hydrogen bonds to make double stranded DNA
- A-T = 2 H-bonds
- C-G = 3 H-bonds
- Between N-H –> O
- N –> H-N
11
Q
What results in sequences of DNA having higher thermal stability
A
- GC rich sequences due to greater number of hydrogen bonds
12
Q
What is Chargaff’s rule
A
- The amount of C=G and A=T
13
Q
Which is the 5’ end
A
- Where sugar is normal orientation- O is pointing up
14
Q
Describe RNA
A
- Similar to DNA but differences
- Single stranded molecule
- Sugar-phosphate backbone contains a ribose sugar
- In RNA the complimentary base to adenine is uracil - unmethylated form of thymine
15
Q
Describe uses of RNA
A
- Cellular organisms use messenger RNA (mRNA) to convey genetic information which directs the synthesis of specific proteins
- RNA is heavily involved in protein synthesis- direct the assembly of proteins on ribosomes
- Protein synthesis uses tRNA to deliver amino acids to the ribosome where rRNA links amino acids together to form proteins
- Carry genetic informations
- RNA viruses (COVID) have genomes composed of RNA
16
Q
Describe structure of RNA
A
- Most biologically active RNAs contain self-complementary sequences that allow parts of the RNA to fold
- H-bonding between complementary sequences leads to secondary structures : hairpin loops, bulges and internal loops
- As RNA is charged, metal ions such as Mg2+ are needed to stabilise many secondary and tertiary structures
17
Q
Define DNA synthesis
A
- DNA synthesis is the natural or artificial creation of DNA molecules
18
Q
What are the 3 main types of DNA synthesis
A
- DNA replication: DNA biosynthesis- in vivo DNA amplification
- PCR: enzymatic DNA synthesis - in vitro DNA amplification
- Gene synthesis: Physically creating artificial gene sequences
19
Q
What are the different enzymes involved in DNA replication
A
- Topoisomerase
- DNA helicase
- Single-stranded binding proteins
- Primase
- Polymerase
- Exonuclease
- Ligase
20
Q
What does topoisomerase do
A
- Relaxes DNA from its supercoiled nature
- DNA gyrase is an example
21
Q
What does DNA helicase do
A
- Separates two strands of DNA by breaking hydrogen bonds
22
Q
What do single-strand binding proteins do
A
- Bind to single-stranded DNA to prevent DNA double helix from re-annealing after DNA helicase unwinds it
23
Q
What does primase do
A
- Provides a starting point for DNA polymerase to begin synthesis of a new DNA strand
24
Q
What does polymerase do
A
- Adds nucleotides to form a new DNA strand
25
What does exonuclease do
1. removes RNA primer
26
What does ligase do
1. Links DNA fragments
27
What direction does polymerase add nucleotides
1. 5' to 3' direction
28
Describe role of DNA polymerase
1. Responsible for catalysing the addition of nucleotide substrates to DNA
2. Polymerases cannot initiate synthesis of new strands but can extend an existing DNA or RNA strand (primer) paired to a template DNA strand
3. Highly accurate
4. Some have proofreading ability- capable of distinguishing mismatches in newly synthesised DNA from original strand sequence
29
What are the 3 steps of DNA biosynthesis
1. Initiation
2. Elongation
3. Termination
30
Describe Initiation
1. AT rich regions called origins are targeted by initiator proteins to form an origin recognition complex
2. Once origin is located a pre-replication complex can be formed which unwinds dsDNA
31
Describe elongation
1. Primase adds primers to allow DNA polymerase to synthesise DNA from 5' to 3' end
32
How is addition of nucleotides different for each strand in DNA
1. Leading strand - continuous
2. Lagging strand 3'-5'- added as okazaki fragments- complementary strand would have to be added 3'-5'
33
Describe why Okazaki fragments are needed
1. The lagging strand is a strand of new DNA whose synthesis is opposite to the direction of the growing replication fork
2. Synthesised in short, separate fragments
34
Describe how Okazaki fragments are added
1. Primase initiates synthesis of a short RNA primer
2. DNA polymerase extends primed segments forming Okazaki fragments
3. RNA primers are removed and replaced with DNA (Exonuclease/DNA polymerase)
4. DNA fragments are joined together by DNA ligase
35
Describe PCR
1. A form of enzymatic DNA synthesis used in the lab
2. PCR is used to amplify a single gene sequence from a segment of DNA
3. Relies on thermal cycling
36
What are the components of PCR
1. DNA template- contains target region to amplify
2. DNA polymerase- use a heat-resistance polymerase- Taq
3. DNA primers- Need two DNA primers that are complementary to the 3' end of each strand of DNA
4. Deoxynucleoside triphosphates- dNTPs- Nucleosides containing triphosphate groups are building blocks for DNA polymerase to synthesise a new strand
37
Where does the reaction take place
1. buffer solution suitable for the enzyme
38
What are the 3 stages of PCR and temps at the stage
1. Denaturation (96 degrees)
2. Annealing (55-65 degrees)
3. Extension (72 degrees)
4. Repeat
39
Describe what happens in denaturation
1. Heat reaction to denature DNA strands to provide a single stranded template for the next step
40
Describe what happens in annealing
1. Cool reaction mixture so primers can bind to their complementary sequences on the single-stranded template
41
Describe what happens in extensino
1. DNA polymerase synthesises a new DNA strand complementary to the DNA strand by adding free complimentary dNTPs from reaction mixture
42
Describe how to use a PCR thermocycler
1. DNA, DNA polymerase, buffer, nucleoside triphosphates and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler
43
What is gel electrophoresis used for
1. To check whether the PCR successfully generated the required DNA target region
2. Agarose gel electrophoresis is used
44
How does gel electrophoresis work
1. Size separation of PCR products by mass and charge
2. THe size of PCR products is determined by comparison of a DNA ladder of known length which is a molecular weight marker
3. The DNA is visualised with a stain (commonly Ethidium bromide) under UV light
45
What are the advantages of PCR
1. Simple technique to understand and use
2. Highly sensitive
3. Produces millions of copies of a specific product which can be used in sequencing, cloning and analysis
46
What are the disadvantages of PCR
1. Prone to contamination
2. Requires information about target sequence in order to generate primers to allow for selective amplification
3. DNA polymerase are prone to error which can lead to mutations in sequences
47
What are applications of PCR
1. Structural analysis
2. DNA typing
3. Disease detection
4. Cloning
5. Mutation analysis
6. Detection of gene expression
7. Mapping
8. site-directed mutagenesis
9. sequencing
