Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Genetic Engineering > cDNA and Genomic Libraries > Flashcards

cDNA and Genomic Libraries Flashcards

1
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

A

Larger gene for expression, cDNAs and small operons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

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

A

For genomic fragments for a genomic library and genomic reconstruction experiments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

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

A

Plasmid-base vectors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Give the types of vectors needed to know?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are essential features of all plasmids?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What requirements of the bacterial host are needed?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Why do you remove 5’ phosphate with alkaline phosphorylase?

A

To prevent vector religation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are advantages of using plasmids as vectors?

A

Good for 1-10kb range

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are the disadvantages of using plasmids as vectors?

A

Limited size limit, poor transformation efficiency.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the genomic organisation of bacteriophage lambda?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the lambda life cycle?

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is lysis in the lambda life cycle?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is lysogeny in the lambda life cycle?

A

Integration of the lambda DNA into the host genome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

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

A

Lysis is a transparent liquid but lysogeny is not.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are plaques?

A

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

18
Q

What plaques arise in lysis and lysogeny?

A

In lysis clear plaques but in lysogeny they are turbid.

19
Q

What is the Lytic pathway?

A

Lambda DNA replication through the rolling circle mechanism

20
Q

Describe the Lytic pathway?

A

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.

21
Q

What constraint occurs dying lambda replication?

A

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.

22
Q

What are possible methods of selection?

A

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

23
Q

What does lacZ complementation provide?

A

A visual identification of recombination clones.

24
Q

What does lacZ encode?

A

Beta- galactosidase

25
Q

How does lacZ complementation work?

A

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.

26
Q

What does the size of a vector chosen depend on?

A

The purpose of cloning and size of insert

27
Q

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

A

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

28
Q

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

A

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.

29
Q

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

A

Lambda insertion vectors

30
Q

What size DNA insert can lambda insertion vectors carry?

A

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

31
Q

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

A

25 %

32
Q

What is a lambda replacement vector?

A

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.

33
Q

Which replacement or insertion vectors have a lower size limit?

A

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

34
Q

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

A

Transformation into E.coli

35
Q

What are cosmids?

A

Plasmids that contain cos sites

36
Q

What can cos sites be used for?

A

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

37
Q

What will in vitro packaging extracts insert?

A

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

38
Q

What do cosmids form on agar plates?

A

Colonies

39
Q

What is bacteriophage M13?

A

A filamentous phage with a single stranded, 6.4 kb genome

40
Q

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

A

Rearrangement.

41
Q

What is the life cycle of M13?

A

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.

42
Q

What are M13 good to make?

A

Genomic libraries but limited by transformation efficiency

Genetic Engineering (10 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions