Week 6

Question 1

What is a genome

Answer 1

The complete genetic material present in a cell

Question 2

What is the transcriptome

Answer 2

Sum of the total mRNA expressed from the genes

Question 3

Proteome

Answer 3

The entire complement of protein produced and expressed by a cell, tissue or organism

Question 4

functional assays

Answer 4

systemic experiments used to explain the function of a protein in a cellular pathway and gene activity

include - flow cytometry,

Question 5

what are the two DNA cloning methods

Answer 5

restriction enzymes
cloning vectors

Question 6

restriction enzymes

Answer 6

used to cleave DNA at specific sites

Question 7

cloning vectors

Answer 7

bacterial plasmids or viruses
to carry inserted foreign fragments

Question 8

what is DNA cloning technique

Answer 8

joining together sequences from different sources
also called recombinant DNA

Question 9

what is genetic engineering

Answer 9

changing genetic content
additions and deletions will be inherited

includes artificial changes in the DNA content
becomes part of the genome

creates a transgenic organism

Question 10

genetic engineering process

Answer 10

1. vector and DNA fragment
2. forms recombinant DNA
3. recombinant DNA replicates within host cells
4. isolation, sequencing and manipulation of purified DNA fragment

Question 11

what are nucleases

Answer 11

• hydrolyse an ester within a phosphodiester bond
• can be dna or rna specific
  needed for specific reassembly of dna fragments

Question 12

what are the nucleases

Answer 12

endonucleases
restriction endonucleases - type II endonucleases
isoschizomers
neoschizomers

Question 13

what are endonucleases

Answer 13

cleave WITHIN a nucleic acid chain - dna or rna

Question 14

what are exonucleases

Answer 14

cleave from either END of dna or rna

Question 15

what are restriction endonucleases

Answer 15

• are bacterial enzymes
• needed for production of recombinant dna
• can cleave double stranded dna at specific sites

example - type II endonucleases

Question 16

what are bacterial enzymes

Answer 16

enzymes found naturally in bacteria

Question 17

what are type II endonucleases

Answer 17

most commonly used restriction endonuclease
same recognition and cleavage site

different from type I as those sites are far from each other

specificity may vary - some might allow similar sequences

Question 18

how can type II restriction endonucleases be cut

Answer 18

can be staggered with sticky ends
- single stranded complementary overlap

blunt double stranded cut
- no overlap
- can still be useful in labs

Question 19

what are isoschizomers

Answer 19

pairs of two different enzymes
both specific to the same recognition site
cut in the same way

Question 20

what are neoschizomers

Answer 20

• different enzymes with the same recognition site
• cut differently can create different sticky ends

Question 21

what is a modification enzyme

Answer 21

produced by bacteria for each restriction enzyme with the same recognition sequence - usually a DNA methyltransferase

as a methylated recognition sequence cannot be cleaved by the restriction enzyme
a bacteriums own genome is protected by modification

Question 22

what is a vector

Answer 22

cloning requires a specifically engineered vector
often as a plasmid
can be used to propagate an incorporated dna sequence in the host

Question 23

what are some features of vectors

Answer 23

ORI, selective marker, engineered multiple cloning site, dna ligase

Question 24

what is an ORI

Answer 24

origin of replication
dna sequence that signals the start

Question 25

what is a selective marker

Answer 25

identifies transformants to allow for growth and eliminates non-transformants

Question 26

what are transformants

Answer 26

cells with foreign genetic material

Question 27

engineered multiple cloning site

Answer 27

a synthetically generated sequence of DNA contains multiple restriction sites series of tandem (two or more) restriction endonuclease sites usd in cloning vectors to create recombinant molecules different enzymes can be chosen to create specific sticky ends

Question 28

what is dna ligase

Answer 28

used for dna fragments with sticky or blunt ends

Question 29

How is bacterial transformation done in the lab

Answer 29

Done by calcium chloride And heat shock Not effective so selective marker is used to collect the recombinant dna that have acquired antibiotic resistance

Question 30

Why is bacterial amplification and purification done

Answer 30

To clear negative bacteria And select the positive bacteria that contain the plasmid

Question 31

How is bacteria containing the plasmid amplified

Answer 31

Colonies are selected and grown in liquid broth containing the antibiotic Negative bacteria dies out and positive bacteria survive and give rise to more colonies

Question 32

How is plasmid dna isolated from the bacteria

Answer 32

Alpine lysis based spin colomn

Question 33

How do false positives of plasmids occur

Answer 33

Plasmid recircularises without a cloned fragment

Question 34

What is blue white selection

Answer 34

Allows bacteria with a vector and insert to be identified

Question 35

How does blue white screening work

Answer 35

The multiple cloning site is in the LacZ operant LacZ produced beta galactosidase which turns blue when X-gal is added So plasmids that contain the dna fragment interrupt beta galactocidase activity - and appear white The white colonies are selected

Question 36

What is a dna library

Answer 36

Collection of dna molecules that are cloned into a vector Effectively represent all dna sequences in the genome

Question 37

Pros/cons of dna library

Answer 37

Useful for representing the genome of simple organisms cDNA is more useful for higher eukaryotes

Question 38

What is cDNA

Answer 38

Synthetic ‘copy’ DNA That has been transcribed from mRNA - only has exons

Question 39

How is cDNA made

Answer 39

- copies of mRNA are collected and synthesised into plasmid vectors MRNA is easily collected by a primer for the polyA tail Enzyme reverse transcriptase is used to synthesise a complementary DNA to each mRNA - makes a single stranded cDNA Which is then converted into a double stranded molecule

Question 40

How is cDNA inserted into a vector

Answer 40

Sticky end or linked is needed Dna pol synthesises a second strand Fragments are protected by methylation at EcoRi sites Synthetic linkers with EcoRI restriction site is added to double strand cDNA

Question 41

What are the limitations of cDNA

Answer 41

Needs to be transformed (put into) E.coli Only represents expressed DNA - exons Silent genes not represented Over expressed genes will be overrepresneted

Question 42

what is screening the cDNA library

Answer 42

required to identify clones with the interested gene

Question 43

what is hybridisation

Answer 43

ability of complementary dna or rna molecule to associate with each other by base pairing can be used to identify specific nucleic acids - when it is of a labelled oligionucleotide

Question 44

what is a probe

Answer 44

a radioactive nucleic acid - dna or rna used to identify a complementary fragment and locate the DNA sequence signal appears over plasmid dna that is complementary to the probe