DNA cloning and modification

Question 1

Give 4 major applications of genetic modification.

Answer 1

1. Protein production, e.g. insulin
2. Research into gene function by creating knock-out mutants
3. Food production, GM crops and animals
4. Gene therapy

Question 2

When was the first recombinant DNA created and by who?

Answer 2

1972, Paul Berg’s group

Question 3

What was the first recombinant DNA a hybrid of?

Answer 3

The SV40 and Lambda viruses

Question 4

Define recombinant DNA.

Answer 4

DNA that has been formed artificially by combining constituents from different organisms.

Question 5

What is the principle of clonal growth and how is it used in recombinant DNA?

Answer 5

Bacteria grow by dividing into exact clones. You insert the recombinant DNA into a plasmid and as the bacteria grow they produce huge volumes of exact copies.

Question 6

Are plasmids essential to bacterial survival?

Answer 6

No.

Question 7

What kind of genes do plasmids contain?

Answer 7

Additional, non-chromosomal DNA.

Question 8

Plasmids usually provide specific functions that allow bacteria to be well-adapted to their environments. Give 3 examples of plasmid function.

Answer 8

1. Nitrogen fixation
2. Antibiotic resistance
3. Virulence (‘the degree of pathogenicity of a microbe’)

Question 9

Plasmids can be transferred between bacteria, true or false?

Answer 9

True, by conjugation.

Question 10

Transmission rate of plasmids between bacteria is high. True or false?

Answer 10

False - rate of transmission is very low at ~1%.

Question 11

What can increase the rate of transmission of plasmids between bacteria? Give 3 examples.

Answer 11

1. Heat shock
2. Electroporation
3. Application of particular chemicals

Question 12

Give an example of a chemical that can make a bacterial cell more receptive to transformation.

Answer 12

Calcium chloride, CaCl2.

Question 13

What was the first transgenic organism and when was it created?

Answer 13

Xenopus DNA was inserted into bacterial plasmids in 1973.

Question 14

Who created the first transgenic organism in 1973?

Answer 14

Cohen and Boyer.

Question 15

What is the basic principle of creating recombinant DNA?

Answer 15

Digest 2 pieces of DNA with the same restriction endonuclease so they have complementary sticky ends, then join them together in ligation.

Question 16

Plasmids contain an origin of replication. What does this mean?

Answer 16

A particular sequence in the plasmid where replication is initiated.

Question 17

Define a multiple cloning site (MCS).

Answer 17

A section of DNA that contains multiple restriction endonuclease recognition sites.

Question 18

What is a polylinker?

Answer 18

Another name for a multiple cloning site.

Question 19

Multiple cloning sites are typical of engineered plasmids. Why?

Answer 19

Restriction sites typically only occur once within a plasmid.

Question 20

Multiple cloning sites are often flanked by promoters/regulators etc. Why?

Answer 20

To regulate endonuclease activity.

Question 21

What do multiple cloning sites allow?

Answer 21

DNA insertion.

Question 22

How many restriction sites per RE are there in each MCS?

Answer 22

1, but there may be others outside the MCS.

Question 23

Marker genes are often inserted into plasmids, for example resistance or fluorescent protein genes. Why?

Answer 23

Allows you to identify which plasmids have been transformed - those that do not display the marker gene have not uptaken the plasmid.

Question 24

What does the LacZ gene encode?

Answer 24

β-galactosidase.

Question 25

What is β-galactosidase?

Answer 25

A hydrolase enzyme that catalyses the hydrolysis of β-galactosidase into monosaccharides.

Question 26

β-galactosidase mutants are missing something. What is it?

Answer 26

An α-peptide at the N-terminus.

Question 27

Define α-complementation.

Answer 27

Whereby the α-peptide is restored in LacZ mutants.

Question 28

The functional β-galactosidase gene has an effect on X-Gal. What is it?

Answer 28

It turns colourless X-Gal blue.

Question 29

What is X-Gal?

Answer 29

A colourless lactose analogue.

Question 30

Why is α-complementation used in transformation experiments?

Answer 30

To see which bacteria have uptaken the gene - the mutants that turn the substrate blue have been transformed back to wild type.

Question 31

Define a bacteriophage.

Answer 31

Viruses that infect bacteria.

Question 32

Explain briefly how bacteriophages work, give 6 steps.

Answer 32

1. Phage attaches to bacteria and injects chromosome 2. Bacterial chromosome is degenerated by phage enymes 3. Phage chromosome is replicated using bacterial machinery 4. Phage genes expressed to produce new phage components 5. Progeny phages assemble 6. Bacterial cell wall split by lysis, progeny phages released

Question 33

What is M13?

Answer 33

A filamentous bacteriophage.

Question 34

How many closely-packed genes does it have for infection and replication?

Answer 34

10.

Question 35

M13 vectors can accommodate large inserts of DNA. True or false?

Answer 35

False - they only have room for very small inserts, up to 3kbp.

Question 36

M13 phage vectors are often engineered to include lacZ markers and MCS. True or false?

Answer 36

True.

Question 37

Lambda phages can accommodate larger inserts than M13 phages. True or false?

Answer 37

True.

Question 38

What is the size of inserts limited by in Lambda phages?

Answer 38

The amount of DNA that can be packaged into a capsid, between 38-51kbp.

Question 39

Define a capsid.

Answer 39

The protein coat of a virus.

Question 40

What is a cosmid?

Answer 40

A hybrid of a plasmid inserted with Lambda phage cos sequences.

Question 41

What do cos sites allow?

Answer 41

Packaging of DNA into phage capsids.

Question 42

Cosmids allow larger inserts than normal plasmids. How big can they be?

Answer 42

Approx. 45kb as opposed to 15kb of normal plasmids.

Question 43

What is an artificial chromosome?

Answer 43

Synthetic chromosomes made of fragments of DNA inserted into a host chromosome.

Question 44

What is the point of an artificial chromosome?

Answer 44

They are introduced into host cells to propagate and can be used to transfect other cells.

Question 45

Give 2 examples of artificial chromosomes.

Answer 45

1. BAC = bacterial artificial chromosome | 2. YAC = yeast artificial chromosome

Question 46

What is the maximum insert size for a BAC?

Answer 46

Approx. 350kbp

Question 47

What is the maximum insert size for a YAC?

Answer 47

Up to 2000kbp.

Question 48

Put in order 6 possible vectors, with number 1 that accommodates the smallest inserts and number 6 with the largest.

Answer 48

1. M13 phage 2. Normal plasmid 3. Lambda phage 4. Cosmids 5. BAC 6. YAC

Question 49

What is a vector in terms of DNA?

Answer 49

A way of transmitting DNA from one organism to another.

Question 50

Give 2 advantages of using bacteria to amplify target DNA.

Answer 50

1. Cheap and self-propagating | 2. Do not need to know the sequence as you do with PCR

Question 51

What is splicing and why does it happen?

Answer 51

The removal of introns (non-coding DNA) from mRNA transcripts. Splicing ensures only the exons of genes are transcribed.

Question 52

Splicing occurs in both eukaryotes and prokaryotes. True or false?

Answer 52

False - it only occurs in eukaryotes as prokaryotes do not contain introns.

Question 53

You cannot properly express eukaryotic DNA in bacteria. Why?

Answer 53

Because it contains introns.

Question 54

How is it possible to overcome the problem that eukaryotic DNA is not properly expressed in bacteria?

Answer 54

Use eukaryotic mRNA to make cDNA using reverse transcriptase, as mRNA has been spliced so the introns have been removed.

Question 55

Where do reverse transcriptases come from?

Answer 55

Retroviruses.

Question 56

Define a DNA library.

Answer 56

A mixture of recombinant DNA clones that represent a significant proportion of the complete genome. Basically so we have a record of all the genes in an organism.

Question 57

DNA libraries can be of 2 types. What are they?

Answer 57

1. Genomic DNA | 2. cDNA (complementary DNA)

Question 58

What is the point of a DNA library?

Answer 58

Used in genetic screening, DNA preparation and gene expression analysis.

Question 59

When generating a DNA library, larger inserts are better. Why?

Answer 59

Because then fewer clones are required, as each one will contain a larger percentage of the whole genome.

Question 60

Complete and partial digests are used in the generation of DNA libraries. Why?

Answer 60

To create overlapping fragments that can be re-aligned during analysis.

Question 61

How do you search a DNA library for a particular gene? Give 2 methods.

Answer 61

1. Make an ssDNA labelled probe that will attach to the sequence of interest 2. Use antibodies labelled with dye

Question 62

Define a microarray.

Answer 62

A set of DNA sequences representing the entire set of genes (not entire genOME) in an organism, arranged in a grid pattern for genetic testing.