Concepts covered last year

Question 1

in vivo

Answer 1

Studies that are in vivo (Latin for “within the living”; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism.

Question 2

in vitro

Answer 2

In vitro (meaning: in the glass) studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called “test-tube experiments”, these studies in biology and its subdisciplines are traditionally done in labware

Question 3

Restriction enzymes

Answer 3

an enzyme produced chiefly by certain bacteria, that has the property of cleaving DNA molecules at or near a specific sequence of bases.

Question 4

Ligation

Answer 4

Ligation may refer to: Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules.

Question 5

Insertional inactivations screens

Answer 5

Insertional inactivation. Insertional inactivation is a technique used in recombinant DNA engineering where a plasmid (such as pBR322) is used to disable expression of a gene. The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence.

Question 6

Blue/white screen

Answer 6

DNA of interest is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal.

Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies.

Question 7

MCS/Polylinker

Answer 7

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites

Question 8

Plasmid vector characteristics

Answer 8

• selectable marker
• MCS
• ori
• reporter gene e.g. lacZ
• promoters (in expression vector)

Question 9

Identify recombinant cells

Answer 9

• blue/white screening

- antibiotic resistance

Question 10

antibiotic resistance

Answer 10

Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth

Question 11

DNA libraries

Answer 11

• collection of all DNA fragments from a starting population

- use recombinant DNA techniques to generate a collection of cloned DNA fragment - a library

Question 12

cDNA library

Answer 12

• total RNA from specific tissue
• copy mRNA to DNA (cDNA)
• ligate cDNA into suitable vector
• introduce recombinant molecules into host

Question 13

cDNA

Answer 13

a DNA that is complementary to a given RNA which serves as a template for synthesis of the DNA in the presence of reverse transcriptase. — called also complementary DNA.

Question 14

gDNA

Answer 14

The DNA residing in chromosomes inside the nucleus, with all the biological information to be transferred to the next generation, is called genomic DNA

Question 15

Plasmids

Answer 15

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell’s chromosomal DNA.

Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes.

Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.

Question 16

Bacteriophage

Answer 16

A bacteriophage is a virus that infects bacteria.

A bacteriophage, or phage for short, is a virus that infects bacteria.

Question 17

Bacteriophage λ

Answer 17

Bacteriophage λ is a temperate phage, which, upon infection of Escherichia coli, enters either the lytic or lysogenic replication pathway. The latter is defined by the integration of the λ prophage within the bacterial host genome, where it is quiescently inherited by daughter cells.

Question 18

temperate vs virulent

Answer 18

Phages that replicate only via the lytic cycle are known as virulent phages while phages that replicate using both lytic and lysogenic cycles are known as temperate phages.

Question 19

Bacteriophage λ insertion vectors

Answer 19

modified λ vector to eliminate common restriction sites and remove part of the non-essential region

Question 20

Bacteriophage λ replacement vectors

Answer 20

modified λ vector to eliminate common restriction sites and remove entire non-essential region

Question 21

Viral particles

Answer 21

A complete virus particle, known as a virion, consists of nucleic acid surrounded by a protective coat of protein called a capsid.

Question 22

Fosmid vectors

Answer 22

hybrid between phage and F-factor plasmid

cos site for phage packaging

ori site for maintenance in bacteria

genes associated with lytic and lysogenic pathway removed

Question 23

BAC vectors

Answer 23

• bacterial artificial chromosomes
• plasmid based on F-factor
• stably maintains 100 to 150kb inserts
• introduced into cells directly - transformation

Question 24

YAC vectors

Answer 24

Yeast Artificial Chromosome vectors

• maintained as mini chromosome within the cell
• different type of selectable marker - encode AA/nucleotide biosynthetic genes
• clones inserts up to ~500kb

Question 25

Library screening by function

Answer 25

• screen based on functional complementation of a mutation in the target gene
• no info about target gene required

Question 26

Library screening by sequence homology

Answer 26

• screen library using nucleic acid hybridisation

- requires sequence from part of the target gene

Question 27

Library screening by protein recognition

Answer 27

• screen an expression library

- requires antibody raised to protein produced by target gene

Question 28

Subcloning

Answer 28

Subcloning is the process of dividing a large DNA fragment carried in a vector into smaller more manageable DNA fragments each carried independently in its own vector.

non-directional cloning

• cloning DNA fragments with the same sticky ends or blunt ends
• inserts in either orientation

directional cloning

• cloning DNA fragments with different sticky ends
• orientation can be determined using particular combination of RE sites
• inserts in only one orientation