cDNA and Genomic Libraries

Question 1

What are small (0.1-1 kb) fragments usually cloned for?

Answer 1

Production or protein in E.coli, promoters or control elements to study regulation.

Question 2

What are intermediate (1 kb-10 kb) fragments usually cloned for?

Answer 2

Larger gene for expression, cDNAs and small operons

Question 3

What are large (10-100 kb) fragments usually cloned for?

Answer 3

For genomic fragments for a genomic library and genomic reconstruction experiments

Question 4

For gene expression and promoter-probing what type of vector is usually used?

Answer 4

Plasmid-base vectors

Question 5

Give the types of vectors needed to know?

Answer 5

Plasmid, bacteriophage lambda, bacteriophage M13, cosmid and phagemid.

Question 6

What are essential features of all plasmids?

Answer 6

Ability to replicate in host cell
Ability to be readily introduced into host cell
Ability to readily insert foreign DNA into vector

Question 7

What requirements of the bacterial host are needed?

Answer 7

Require efficient transformation by plasmid DNA
Require stable maintenance of transformed DNA
Require disablement of the host for safety reasons

Question 8

What essential features do plasmids have for use as a vector?

Answer 8

Ori, selectable marker, suitable region for insertion of foreign DNA- restriction enzyme sites, suitable size

Question 9

Why do you remove 5’ phosphate with alkaline phosphorylase?

Answer 9

To prevent vector religation

Question 10

What are advantages of using plasmids as vectors?

Answer 10

Good for 1-10kb range

Question 11

What are the disadvantages of using plasmids as vectors?

Answer 11

Limited size limit, poor transformation efficiency.

Question 12

What is the genomic organisation of bacteriophage lambda?

Answer 12

Has a 49 kb linear double stranded DNA which has single stranded complementary 12 nucleotide terminals called cohesive ends.

Question 13

Describe the lambda life cycle?

Answer 13

1. Phage particle attaches to an E.coli cell and injects its DNA
2. The lambda DNA once inside circulates due to its cohesive ends
3. Lysis of lysogeny occurs

Question 14

What is lysis in the lambda life cycle?

Answer 14

Lambda DNA replication, synthesis of phage products (capsid proteins), particle assembly, cell lysis and phage release

Question 15

What is lysogeny in the lambda life cycle?

Answer 15

Integration of the lambda DNA into the host genome

Question 16

What does lysis and lysogeny look like in a liquid culture assay?

Answer 16

Lysis is a transparent liquid but lysogeny is not.

Question 17

What are plaques?

Answer 17

Areas of dead cells that appear clear against the bacterial growth.

Question 18

What plaques arise in lysis and lysogeny?

Answer 18

In lysis clear plaques but in lysogeny they are turbid.

Question 19

What is the Lytic pathway?

Answer 19

Lambda DNA replication through the rolling circle mechanism

Question 20

Describe the Lytic pathway?

Answer 20

When lambda genomes circularise the sites where the cohesive ends join are called cos sites. Replication occurs and the linear DNA is rolled off with individual cos sites encoded. These cos sites are cleaved in the concatenate and packed into a prehead which then associated with preformed tails.

Question 21

What constraint occurs dying lambda replication?

Answer 21

The packaging constraint: only a precise length of DNA can be packaged into the head. This is determined by the cos site and specific cleavage.

Question 22

What are possible methods of selection?

Answer 22

Differential antibiotic resistance, lacZ complementation, Spi selection, cl selection.

Question 23

What does lacZ complementation provide?

Answer 23

A visual identification of recombination clones.

Question 24

What does lacZ encode?

Answer 24

Beta- galactosidase

Question 25

How does lacZ complementation work?

Answer 25

An inactive portion of the promoter is encoded in the vector, and the vector also encodes the regulatory sequences that repress the expression unless IPTG is present. Therefore if you transform with IPTG and the vector is present beta-galactosidase is produced which is blue, and if you insert the DNA of interest into this poly linker and transform the plaques would be white. Therefore white is successful and blue is not.

Question 26

What does the size of a vector chosen depend on?

Answer 26

The purpose of cloning and size of insert

Question 27

Due to the packaging constraint of lambda what size fragments of DNA can be inserted?

Answer 27

Only DNA molecules whose length is 78-105% that of wild-type lambda, which is 48 kb, may be packaged into phage head. Lambda gt10 can except upto 9 kb Other Lambda can accept up to 22 kb

Question 28

How can you overcome the packaging constraint and make lambda accept larger inserts?

Answer 28

The non-essential region of lambda DNA is not required as it only contains proteins for lysogeny integration and excision, removal of this 20 kb fragment can provide space for more insert.

Question 29

What is the name given to bacteriophage lambda who have had their non-essential region removed?

Answer 29

Lambda insertion vectors

Question 30

What size DNA insert can lambda insertion vectors carry?

Answer 30

38-40 kb, depending on how much non-essential region is removed.

Question 31

How much wild type lambda can you delete when making an insertion vector?

Answer 31

25 %

Question 32

What is a lambda replacement vector?

Answer 32

When the non-essential region of the lambda is removed but replaced with a stuffer region which is removed by restriction digest at multiple sites in order to then insert the DNA of interest.

Question 33

Which replacement or insertion vectors have a lower size limit?

Answer 33

Replacement; they are generally designed to carry larger DNA insert of up to 22 kb, and it is not useful for cloning small molecules.

Question 34

What are bacteriophage lambda a lot better at doing in comparison to plasmid based vectors?

Answer 34

Transformation into E.coli

Question 35

What are cosmids?

Answer 35

Plasmids that contain cos sites

Question 36

What can cos sites be used for?

Answer 36

Insertion of over 22 kb that lambda replacement vectors allow for.

Question 37

What will in vitro packaging extracts insert?

Answer 37

Any molecule that carries cos sites separated by 37-50 kb.

Question 38

What do cosmids form on agar plates?

Answer 38

Colonies

Question 39

What is bacteriophage M13?

Answer 39

A filamentous phage with a single stranded, 6.4 kb genome

Question 40

What are large inserts in M13-based vectors prone to?

Answer 40

Rearrangement.

Question 41

What is the life cycle of M13?

Answer 41

The M13 phage recognises and binds F pilus (male specific), the DNA is internalised and transformed from single stranded to double stranded replicative form, this is them replicated and packaged as single stranded DNA into mature phage.

Question 42

What are M13 good to make?

Answer 42

Genomic libraries but limited by transformation efficiency