DNA technology

Question 1

Define genome

Answer 1

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

Question 2

What are the 4 basic genetic engineering tools?

Answer 2

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

Question 3

What are the 5 basic steps of gene cloning?

Answer 3

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

Question 4

Why is gene cloning important?

Answer 4

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

Question 5

What 4 things does PCR require?

Answer 5

ds DNA template
Pair of oligonucleotide primers
DNA polymerase
Nucleotides

Question 6

Why is Taq DNA polymerase used?

Answer 6

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

Question 7

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

Answer 7

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)

Question 8

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

Answer 8

Doubled

Question 9

What are the limitations of PCR?

Answer 9

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

Question 10

What is special about cycle 35?

Answer 10

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

Question 11

Why is less than 10,000 base pairs used?

Answer 11

Easily purified

Avoids DNA breakdown

Question 12

What are plasmid vectors derived from?

Answer 12

Derived from Plasmids found in bacteria

Question 13

What does Ori mean?

Answer 13

Ori- origin of replication

Question 14

What are restriction enzymes used for?

And how do they work?

Answer 14

Cut DNA by hydrolysing phosphodiester bonds

as they recognise specific sequences of DNA bases

Question 15

Describe Host-controlled resistance

Answer 15

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

Question 16

Why don’t restriction enzymes cleave bacterial DNA?

Answer 16

Contains an ECO RI methylase - Methylates the sequence GAATTC

Bacteria dont have this

Question 17

DNA methylate selectively methylate’s what?

Answer 17

Hemi-methylated DNA

Question 18

Define Exonucleases

Answer 18

Removing nucleotides from the ends

Question 19

Define Endonucleases

Answer 19

Break nucleic acid chains in the interior

Question 20

Explain what Type I,II and III of endonucleases do

Answer 20

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

Question 21

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

Answer 21

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

Question 22

Describe how to do gel electrophoresis

Answer 22

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.

Question 23

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

Answer 23

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

Question 24

What are the two types of ligation?

Answer 24

Blunt end and sticky ends

Question 25

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

Answer 25

True

Question 26

True or false you can use nucleases to create blunt ends

Answer 26

True

Question 27

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

Answer 27

True

Question 28

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

Answer 28

BamHI site

Question 29

What are the 5 options to construct a recombinant DNA molecule

Answer 29

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

Question 30

What is TA cloning?

Answer 30

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

Question 31

What is transformation?

Answer 31

Introduction of DNA into living cells

Question 32

What are the 3 steps in transformation

Answer 32

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

Question 33

Define selection

Answer 33

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

Question 34

Transformation is inefficient. Why?

Answer 34

Transformation is an inefficient process. - Only a small % of plasmids are taken up. - Approximately 0.01% of cells take up DNA.

Question 35

Define Tetr and Ampr

Answer 35

``` Ampr= Ampicillin resistance gene Tetr= tetracycline resistance gene ```

Question 36

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

Answer 36

Replica plating (pBR322 vectors) Blue/white selection (pUC vectors) Restriction mapping (any vector)

Question 37

Describe replica plating What is the marker? and what is its role?

Answer 37

Tetracycline marker | Helps us distinguish between recombinant and self ligated vectors

Question 38

Describe Blue/white selection Which gene is used and what does it code for

Answer 38

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

Question 39

What are the 4 steps of restriction mapping?

Answer 39

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

Question 40

To produce protein from a cloned gene you need what?

Answer 40

An expression vector

Question 41

What do expression vectors provide?

Answer 41

Provide transcription and translation.

Question 42

Expression vectors. What catalyses transcription and how? What happens in bacteria

Answer 42

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).

Question 43

Why choose bacteria or animal cells as host cells

Answer 43

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

Question 44

Factor Viii is produced in animal cells | What does it play a central role in?

Answer 44

Plays a central role in blood clotting | Commonest form of haemophilia results from inability to synthesise Factor VIII

Question 45

What is a bioreactor?

Answer 45

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