In Vivo cloning

Question 1

In vivo gene cloning

Answer 1

Isolation and amplification of an individual gene sequence by insertion of that sequence into an organism where it can be replicated.

Question 2

Recombinant DNA

Answer 2

The product of joining together DNA from different sources

Question 3

Uses for DNA cloning

Answer 3

• Genes can be isolated and identified
• Control sequences of DNA can be identified and analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified eg: gene defeats related to specific diseases
• Organisms can be ‘engineered’ for specific purposes eg: insulin production, insect resistance.

Question 4

Restriction enzymes (RE)

Answer 4

• Prevent invasion of foreign DNA (such as viral DNA) by cutting them.
• they recognize a specific DNA sequence (between 4-12bp) which is two fold symmetry and cut both DNA strands
• Depending on the enzyme they can either create staggered ends or blunt ends.
• only found in prokaryotes

Question 5

Endonucleases

Answer 5

Attach foreign DNA from the ends, breaking them down bit by bit.
There are ubiquitous (found everywhere)

Question 6

Methylation

Answer 6

A method of protection by bacteria to prevent RE’s from damaging own DNA.
The methylase enzyme adds a methyl group to DNA at specific sites to protect the site from restriction endonuclease cleavage.
This aids in the identification of foreign DNA to its own (foreign DNA lacks the methylated group)

Question 7

Ligase

Answer 7

Where a restriction enzyme cuts the DNA, Ligase joins two DNA strands together.

Question 8

Cloning Vector

Answer 8

A small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes

Question 9

Vector Features

Answer 9

• Must contain a replicon (origin of replication) that enables it to replicate in host cells
• several marker genes (used to determine if a nucleic acid sequence has been successfully inserted into an organism’s DNA)
• Unique cleavage sites
• For expression must contain control elements, such as promoters, terminators, ribsosome binding sites

Question 10

Types of vectors

Answer 10

• Plasmids
• Phages
• Cosmids
• Yeast Artificial Chromosomes (YACs)
• Bacterial Artificial Chromosomes (BACs)
• Transposons

Question 11

Plasmids

Answer 11

• Naturally occurring extrachromosomal DNA
• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends of plasmid.
• can replicate independently of the host cell.
• Size ranges from a few kb to near 100kb

Question 12

Cloning a gene in a bacterial plasmid

Answer 12

1) Vector (Bacteria containing ampicillin resistance and the human DNA containing gene of interest are cut using the same restriction enzymes.
2) Mixing the two they join by base pairing with some now containing the gene of interest
3) DNA ligase is used to covalently bond the DNA
4) plasmid is plasmid into lacZ bacteria via transformation
5) cells are cloned on a medium containing ampicillin and X-gal
6) identify clones with recombinant plasmid by the ability to grow in ampicillin and their white colour.

Question 13

Why is the gene of interest inserted into the LacZ gene

Answer 13

Adding them into this gene makes it non functional so the bacteria is unable to breakdown lactose - this allows the plasmids containing the gene of interest to be identified. As the functional LacZ gene turns colonies blue in the presence of X-gal.

Question 14

DNA can be inserted into a Cell by:

Answer 14

• Transformation
• Electroporation
• protoplast fusion
• microinjection
• gene gun

Question 15

Three conformations of plasmid DNA

Answer 15

• supercoiled: often referred to as covalently closed circular DNA, ccc
• open-circular (oc),
• linear

Question 16

Which conformation runs fastest?

Answer 16

• Supercoiled - due to its compact natures suffers less friction through agarose
• Linear is second as it travels end first

Question 17

Expressing eukaryotic genes in bacteria

Answer 17

• Eukaryotic DNA contains introns (intervening sequences) and exons (expressed or translated sequences), so to express the eukaryotic gene introns are edited out after mRNA transcription

Question 18

Disadvantages of using bacterial host cells

Answer 18

May not be able to use the information in a eukaryotic gene due to the use of different enzymes.
Unable to make the post-translational modifications required to produce some eukaryotic proteins

Question 19

Problem: Eukaryotic genes of interest may be two large to clone easily because they contain long noncoding regions (introns), which prevent correct expression of the gene by bacterial cells, which lack RNA-splicing machinery.

Answer 19

Solution: Scientists can make artificial eukaryotic genes that lack introns.
Artificial chromosomes, which combine the
essentials of a eukaryotic chromosome with foreign DNA, can carry much more DNA than plasmid vectors, thereby enabling very long pieces of DNA to be cloned.

Question 20

Cosmids

Answer 20

• Plasmid vectors that contain a bacteriophage lamda cos site
• The cos site results in efficient packaging of lamda DNA into virus particles
• So, with the cos site, larger DNA inserts
  are possible (up to ~40 kb)

Question 21

Cosmid cloning Vectors

Answer 21

• Fragments from 30 to 46 kb
• Cosmids combine essential elements of a
plasmid and Lambda systems.
• Cosmids are extracted from bacteria and
mixed with restriction endonucleases.
• Cleaved cosmids are mixed with foreign
DNA that has been cleaved with the same
endonuclease.
• Recombinant cosmids are packaged into
lambda caspids
• Recombinant cosmid is injected into the
bacterial cell where the r cosmid arranges
into a circle and replicates as a plasmid

Question 22

YACs

Answer 22

• Yeast Artifical Chromosome
• Artifically produced mini chromosome
• They contain a yeast centromere, two yeast teleomeres, bacterial origin of replications and marker genes
• Can accomodate large inserts (1,000 to 2,000kb)

Question 23

Purpose of YACs

Answer 23

YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.

Question 24

DNA library

Answer 24

• A genomic library is a collection of many bacterial or phage clones, each containing copies of a particular DNA segment from a foreign genome.
• However some genes may be divided up between two or more genes.

Question 25

Phage Cloning Vectors

Answer 25

• Fragments up to 23 kb can be may be accommodated by a phage vector
• Lambda is most common phage
• 60% of the genome is needed for lyticpathway.
• Segments of the Lambda DNA is removed and a stuffer fragment is put in.
• The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector.

Question 26

Examples of Phage Cloning Vectors

Answer 26

• Example: Charon4A Lambda -When Charon4A Lambda is intact, beta-galactosidasereacts with X-gal and the colonies turn blue.
• When the DNA segment replaces the stuffer region, the lac5 gene is missing, which codes for beta-galactosidase, no beta-galactosidaseis formed, and the colonies are white.