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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

DNA polymerase 3-

Multimeric enzyme in which specific properties are contributed by different subunits

Makes tRNAs