3.4.10 Recombinant DNA Technology Flashcards Preview

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Flashcards in 3.4.10 Recombinant DNA Technology Deck (58)
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1

What is recombinant DNA?

DNA from 2 different sources are combined

2

What does the production of DNA fragments involve?

Transferring a fragment of DNA from one organism to another

3

Explain why the recipient and donor organisms don’t have to be same species when transferring a fragment of DNA from one organism to another

  • ∵ genetic code is universal
    • (same DNA base triplets code for same amino acids in all living things)
  • ∵ transcription and translation mechanisms are pretty similar 

4

Name 3 methods of making DNA fragments

  • Using Reverse Transcriptase
  • Using Restriction Endonuclease Enzymes
  • Using a 'Gene Machine'

5

Why is mRNA easier to obtain than DNA?

Many mRNA molecules (complementary to a gene)

6

What does reverse transcriptase do?

Makes DNA from an RNA template

7

What is the DNA produced by reverse transcriptase called?

complementary DNA (cDNA)

8

Describe how you can make DNA fragments using reverse transcriptase

  1. mRNA is isolated from cells
  2. Its mixed with free DNA nucleotides and reverse transcriptase
  3. Reverse transcriptase uses mRNA as a template to synthesis a new strand of cDNA
  4. DNA polymerase is used to build up the complementary base pairings with cDNA = DNA is formed

9

Some sections of DNA have ________ sequences of nucleotides

palindromic

10

What are palindromic sequences of nucleotides?

Sequences that consist of antiparallel base pairs

(Base pairs that read the same in opposite directions)

11

What are restriction endonucleases?

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) DNA at these places

12

Why do different restriction endonucleases cut at different specific recognition sequences?

∵ shape of recognition sequence is complementary to enzyme's active site

13

When can you use restriction endonucleases to create a DNA fragment?

 If recognition sequences are present either side of DNA fragment

14

Describe how you can use restriction endonuclease to produce a DNA fragment

  1. DNA sample is incubated with specific restriction endonuclease & it cuts DNA fragment out via hydrolysis reaction
  2. Sometimes cut leaves sticky ends - small tails of unpaired bases at each end of the fragment
  3. Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences

15

What is meant by a 'gene machine'?

  • Technology that enables fragments of DNA that can be synthesised from scratch without need for pre-existing DNA template
    • Database contains necessary info to produce DNA fragments

16

What does a 'gene machine' allow you to do?

Can produce DNA sequences that don't exist naturally

17

Describe how you can use a 'gene machine' to produce a DNA fragment

  1. Sequence required is designed
  2. 1st nucleotide in sequence is fixed to some sort of support
    • e.g. a bead
  3. Nucleotides are added step by step in correct order, in a cycle of processes that include adding protecting groups
  4. Short sections of DNA (called oligonucleotides - 20 nucleotides long) are produced
  5. Once complete, they're broken off from support and all protecting groups are removed
  6. Oligonucleotides can then be joined together to make longer DNA fragments

18

Using a 'Gene Machine' to Producing DNA Fragments

Explain why protecting groups are added when nucleotides are added step by step in correct order

Protecting groups make sure nucleotides are joined at the right points, to prevent unwanted branching

19

Name 2 methods of amplifying (makes lots of copies of) DNA fragments

  • In Vivo Amplification
  • In Vitro Amplification

20

Name the 3 stages in in vivo amplification

  1. DNA Fragment is Inserted into Vector
  2. Vector Transfers DNA Fragment into Host Cells
  3. Identifying Transformed Host Cells

21

In Vivo Amplification

What vectors are normally used?

Can be plasmids or bacteriophages (viruses that infect bacteria)

22

What is a recombinant plasmid?

When a gene is added from another organism to the plasmid 

23

In Vivo Amplification

1) Describe how a DNA fragment is inserted into a vector

  1. Vector DNA is cut open using same restriction endonuclease that was used to isolate DNA fragments containing the target gene
    • So sticky ends of vector are complementary to sticky ends of DNA fragment containing the gene
  2. Vector DNA and DNA fragment are mixed together with DNA ligase
    • DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA
      • aka ligation

24

Describe how a plasmid vector transfers recombinant DNA into host cells

Host cells are to be persuaded to take in the plasmid vector and its DNA

25

Describe how a bacteriophage vector transfers recombinant DNA into host cells

  • Bacteriophage infect host bacterium by injecting its DNA into it
  •  Phage DNA (with the target gene in it) then integrates into bacterial DNA

26

Host cells that take up vectors containing the gene of interest are said to be ______

transformed

27

What are used to identify transformed cells?

Marker genes

28

When are marker genes inserted into vectors and why?

  • Inserted at same time as target gene (i.e. gene to be cloned)
  • ∴ transformed host cells will contain target gene and marker gene

29

What are host cells grown on?

Agar plates

Each cell divides and replicates its DNA, creating colony of cloned cells

30

Name 3 examples of marker genes

  • Genes that code for antibiotic resistance
  • Genes that code for fluorescence
  • Enzyme markers

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