Flashcards in DNA structure and replication Deck (44)
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1
What evidence is there that DNA is the genetic material?
Griffith, 1928:
Studied the transformation of pneumococci in vivo = information can be transferred between different strains of bacteria
Hershey & Chase, 1952:
Studied bacteriophage infection
= DNA can be transferred to infect other bacteria = DNA is the genetic material not protein
2
Phage =
Virus that infects bacteria and multiply inside the cells causing lysis which releases progeny phage to infect other bacteria
3
Purine =
- Larger
- Composed of 2 fused rings incorporating 2 nitrogen atoms in each ring
- Adenine and Guanine
4
Pyrimidines =
- Smaller
- Single ring with 2 nitrogen atoms
- Thymine, Cytosine, Uracil
5
Nucleotide =
Nucleoside + Phosphoric acid
6
Nucleoside =
Nitrogen base + Pentose sugar
7
Central Dogma
DNA > transcription > RNA > translation > Protein
8
Enzymes that manipulate DNA
- Nucleases
- Ligases
- Polymerases
- Kinases + phosphatases
- Methylases + demethylases
- recombinases + topisomerases
9
When DNA is melted …
Two strands are separated by thermal vibration - disrupts hydrogen bonds that holds strands together
10
Anneal/hybridise =
DNA strands joined back together during slow cooling
11
Endonuclease -
Cuts double stranded DNA at internal sites
= sticky ends (staggered cut) or blunt ends (straight cut)
12
Exonucleases -
Cuts DNA strands at the end
13
Ligate =
Join together two DNA fragments that have been cut by the same enzyme = recombinant DNA
14
Restriction endonucleases =
- enzymes used to generate DNA fragments for subsequent re-assortment, ligation and cloning
- recognise + chop DNA
- protects microorganisms from foreign DNA by recognising inverted repeats (palindromes) because they bind to DNA as diners
15
Example of an restriction endonuclease…
Thermostable DNA polymerase is used in the polymerase chain reaction
16
Polymerase chain reaction =
An artificial method for multiplying fragments of DNA
Makes many copies of a specific sequence of DNA in vitro
17
To carry out the polymerase chain reaction you need…
-DNA primers
-Taq polymerase
-free nucleotides
-Buffer
18
Steps of the polymerase chain reaction…
1) denaturation - 96 degrees Celsius = splits DNA into single strands
2) annealing - 55 degrees Celsius = primers bind to single strand, this creates a small section of double stranded DNA
3) extension - 72 degrees Celsius = taq polymerase binds to double stranded DNA and adds free nucleotides
19
Gene cloning =
Isolating DNA from one organism and propagating it in another organism to produce a genetically identical copy
20
Cloning vector =
A small piece of DNA, taken from a virus/plasmid that has a foreign DNA fragment inserted in it - can insert DNA into a host cell
21
Plasmid =
Small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently
22
Ligation =
The joining of two DNA strands by a phosphate Ester linkage
- used to form circular recombinant plasmids
23
Transformation =
Alteration of the genetics of a cell by the direct uptake and expression of DNA from its surroundings
- how recombinant plasmids are introduced into host cells
24
Sanger Method of gene cloning…
-gene that is being sequenced is cut out of the DNA strand using restriction enzymes
-gene is inserted into a vector (plasmid)
-plasmid is taken up by bacterium
-when bacteria divides by binary fission the plasmid with the gene is copied
25
Meselson & Stahl, 1958…
…Proved that DNA replication proceeds via a semi-conservative mechanism using density gradient centrifugation
- used light Nitrogen-14 and heavy Nitrogen-15 which form separate bands at positions corresponding to their buoyant densities.
-found that the strands separate and each act as a template for synthesis
26
Requirements of DNA polymerase 1…
-dNTPs plus Mg2+
-template
-primer with 3’-OH
27
All DNA polymerases…
-require dNTPS plus Mg2+, DNA templates and primers
-synthesise in the 5’ > 3’ direction
-have 3’ > 5’ exonuclease activity
-processive
28
Differences between DNA polymerases…
-polymerase 1 and 2 are slow but polymerase 3 is faster
29
DNA polymerase 1 -
A single polypeptide chain with 3 structural domains
Makes large rRNAs
30
