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BIOL1024 > DNA technology > Flashcards

DNA technology Flashcards

1
Q

Define genome

A

the length of DNA in 1 haploid set of chromosomes entire gene set

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2
Q

What are the 4 basic genetic engineering tools?

A
  1. Plasmid vectors
  2. Restriction enzymes to cut DNA
  3. Gel electrophoresis to separate DNA fragments
  4. DNA ligase to join DNA fragments
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3
Q

What are the 5 basic steps of gene cloning?

A
  1. The gene is isolated
  2. Gene is inserted into vector
  3. Transported into a host cell
  4. Multiplication of recombinant DNA
  5. Division of host cell colony
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4
Q

Why is gene cloning important?

A

To generate large amounts of DNA
To generate large amounts of desired protein
To detect mutations in gene sequencing

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5
Q

What 4 things does PCR require?

A

ds DNA template
Pair of oligonucleotide primers
DNA polymerase
Nucleotides

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6
Q

Why is Taq DNA polymerase used?

A

Taq DNA Polymerase has a temperature optimum of 72°C and is relatively stable at 94°C compared to early polymerases

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7
Q

What are the 3 steps in the PCR that require different temp and what are they?

A

Step 1 – Denature template DNA to ssDNA with heat (95)
Step 2 – Lower temperature to allow primers to anneal (55)
Step 3 – DNA Polymerase extends primer - Go to step 1 (72)

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8
Q

Every cycle of Polymerase Chain reaction the total DNA template is ……..

A

Doubled

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9
Q

What are the limitations of PCR?

A

: Difficult to amplify products longer than 10-15kb. : Need to know the sequence flanking your gene

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10
Q

What is special about cycle 35?

A

Increasing the cycle number above 35 has little positive effect: reagents are depleted
: polymerase is damaged

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11
Q

Why is less than 10,000 base pairs used?

A

Easily purified

Avoids DNA breakdown

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12
Q

What are plasmid vectors derived from?

A

Derived from Plasmids found in bacteria

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13
Q

What does Ori mean?

A

Ori- origin of replication

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14
Q

What are restriction enzymes used for?

And how do they work?

A

Cut DNA by hydrolysing phosphodiester bonds

as they recognise specific sequences of DNA bases

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15
Q

Describe Host-controlled resistance

A

Phage injects DNA into bacterium
Restriction enzymes bind to DNA from phage
Phage DNA is cleaved and inactivated

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16
Q

Why don’t restriction enzymes cleave bacterial DNA?

A

Contains an ECO RI methylase - Methylates the sequence GAATTC

Bacteria dont have this

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17
Q

DNA methylate selectively methylate’s what?

A

Hemi-methylated DNA

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18
Q

Define Exonucleases

A

Removing nucleotides from the ends

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19
Q

Define Endonucleases

A

Break nucleic acid chains in the interior

20
Q

Explain what Type I,II and III of endonucleases do

A

Type I- cuts DNA 1000 bases from recognition site
Type II- cuts DNA at recognition site
Type III- cuts DNA 25 bases from recognition site

21
Q

Describe the nucleophilic attack that occurs when RE break a phosphodiester bond

A

Arrow from Nucleophile B to H from H20
Arrow from O in H20 to P in phosphate
Arrow from P-O to H-A
Arrow from bond between H-A to A

Leaves a O- on phosphate

22
Q

Describe how to do gel electrophoresis

A

a) Separation of DNA fragments by gel electrophoresis

b) Visualising DNA in a gel with nucleic acid binding dyes and UV light e.g. Ethidium bromide or Gelred.

23
Q

Describe how a gene gets added to a plasmid// form recombinant DNA

A

Requires enzyme DNA ligase (Usually T4 DNA ligase)
Which catalyses a phosphodiester bond
And requires ATP

24
Q

What are the two types of ligation?

A

Blunt end and sticky ends

25
Q

True or false you can cut the vector and gene with the same and different RE

A

True

26
Q

True or false you can use nucleases to create blunt ends

A

True

27
Q

True or false you can use DNA pol to fill in ends

A

True

28
Q

Name a typical linker you can use to join vector and the desired gene

A

BamHI site

29
Q

What are the 5 options to construct a recombinant DNA molecule

A
  1. Cut vector and gene w/ same RE
  2. Cut with different
  3. If gene and vector dont have compatible enzymes use nucleases to create blunt ends and DNA pol to fill the ends
  4. Use linkers and adaptors
  5. Use TA cloning
30
Q

What is TA cloning?

A

Adds a single 3’ adenine overhang to each end by adding thymine and terminal transferase

31
Q

What is transformation?

A

Introduction of DNA into living cells

32
Q

What are the 3 steps in transformation

A
  1. Prepare competent bacterial cells
    • limited normal uptake
    • chemical/physical treatment
  2. Introduce DNA into cells at 50mM CaCl2 treatment
  3. Select those cells that have taken up the recombinant DNA via 42. 2 min heat shock
33
Q

Define selection

A

most vectors confer antibiotic resistance which helps determine which cells harbour the recombinant DNA molecule. (how we determine which have taken up the gene

34
Q

Transformation is inefficient. Why?

A

Transformation is an inefficient process.

- Only a small % of plasmids are taken up.
- Approximately 0.01% of cells take up DNA.
35
Q

Define Tetr and Ampr

A 
Ampr= Ampicillin resistance gene
Tetr= tetracycline resistance gene
36
Q

What are the 3 ways we can distinguish bacteria that have taken up the recombinant DNA or self ligated vector?

A

Replica plating (pBR322 vectors)

Blue/white selection (pUC vectors)

Restriction mapping (any vector)

37
Q

Describe replica plating

What is the marker? and what is its role?

A

Tetracycline marker

Helps us distinguish between recombinant and self ligated vectors

38
Q

Describe Blue/white selection

Which gene is used and what does it code for

A

Lac z gene encodes for Enzyme beta galactosidase - selectable marker

Beta galactosidase converts lactose into galactose and glucose

X-gal is an analog of lactose and turn blue when cleaved by Beta galactosidase

39
Q

What are the 4 steps of restriction mapping?

A

Transformation ligation mix into bacteria
Plate out on agar + ampilcillin
Grow up bacteria and extract DNA
Using restriction enzymes to distinguish between recombinant and self ligated vector

Recombinant DNA has more markers

40
Q

To produce protein from a cloned gene you need what?

A

An expression vector

41
Q

What do expression vectors provide?

A

Provide transcription and translation.

42
Q

Expression vectors.
What catalyses transcription and how?

What happens in bacteria

A

Transcription is catalysed by RNA polymerase

RNA pol binds to the promoter region of a gene

In bacteria - ribosome attaches to the mRNA via a ribosome binding site (rbs).

43
Q

Why choose bacteria or animal cells as host cells

A

i) Bacteria - Grow in liquid media
- Divide every 20mins
- Bacteria do not glycosylate proteins
ii) Animal cells - Need to grow attached to a solid matrix
- Divide every 18 hours
- will glycosylate proteins

44
Q

Factor Viii is produced in animal cells

What does it play a central role in?

A

Plays a central role in blood clotting

Commonest form of haemophilia results from inability to synthesise Factor VIII

45
Q

What is a bioreactor?

A

Animals are bioreactors which have foreign gene inserted into them. eg spider silk in milk

BIOL1024 (14 decks)

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