Recombinant DNA technology

Question 1

What is the central dogma?

Answer 1

The flow of DNA to protein, via mRNA

Question 2

What is replication?

Answer 2

using DNA polymerase to make identical DNA molecules

Question 3

What is transcription?

Answer 3

using RNA polymerase to make RNA is using DNA as a template.

Question 4

What is translation?

Answer 4

where proteins are made using mRNA

Question 5

What is the start codon?

Answer 5

Methionine ATG

Question 6

What is a gene?

Answer 6

consists of regulatory domains and an open reading frame that contains exons and introns.

Question 7

What are regulatory domains?

Answer 7

Promoter - tells the RNA polymerase excatly where to start trasncribing
Activator
Silencer
Terminator - where transcription stops

Question 8

How do we know the genes are eukaryotic?

Answer 8

Eukaryotic genes have exons and introns
Prokaryotes only have introns

Question 9

What is the difference between a start codon and a promoter?

Answer 9

start codon comes directly after the promoter.
Start codon - initiates translation
Promoter - initiates transcription

Question 10

How can we manipulate DNA?

Answer 10

Using restriction enzymes, DNA ligase and plasmids.

Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites = makes many cuts resulting in restriction fragments
Most useful enzymes cut the molecule in a staggered way producing sticky ends that bind to the complementary sticky ends of other fragments.

DNA ligase seals the bonds between fragments.

Question 11

What is plasmid?

Answer 11

Small circular piece of DNA that can replicate independently of the chromosome.

Question 12

What is an antibiotic resistance marker?

Answer 12

Identify organisms that contain this plasmid

Question 13

What is the origin of replication?

Answer 13

DNA sequence where plasmid replication starts.

Question 14

What is the MCS?

Answer 14

Mulitple cloning site
a short DNA sequence containing many unique restriction enzyme-cutting sites for different options for cloning the DNA of interest into the plasmid.

Question 15

WHat is an insert?

Answer 15

Pieces of DNA that have ligated into the plasmid, in the MCS

Question 16

What is the promoter region?

Answer 16

Drives transcription of insert DNA
Leads to the production of the recombinant DNA protein.
Also can lead to constitutive expression, expression at certain growth phases or be induced by chemicals.

Question 17

What is a selectable marker?

Answer 17

Allows selection of an organism that contains that piece of DNA
Bacteria = antibiotic resistance gene
plants = herbisicde resistance gene

Question 18

What is the primer binding site?

Answer 18

Short piece of DNA where synthesized DNA primers can bind, either to allow sequencing of the DNA or to amplify the polymerase chain reaction.

Question 19

Steps in recombinant DNA tech

Answer 19

Restriction enzyme cuts sugar phosphate backbones
DNA fragment is added from another molecule cut by the same enzyme
Base pairing occurs
Only one possible combination = ensures that is is identical when flipping it around.
DNA ligase seals the strand.

Question 20

What is DNA-cloning?

Answer 20

Multiplication of a DNA fragment using bacteria
Cut plasmid in the MCS with EcoR1
Cut chromosome with EcoR1
Ligase the plasmid together with the isolated DNA strand.
INsert plasmid into bacteria
Innoculate bacteria in media and incubate.
Use chemical to isolate DNA , end up with much larger amount of DNA than when started.

Question 21

what is a problem of bacterial cloning?

Answer 21

Identifying the bacteria containing plasmids with an insert. An antibiotic can select for bacterium that contains a plasmid.

Question 22

How can we tell if a bacterium contains a plasmid with an insert or not?

Answer 22

a-complementation:
E.coli has modified strands of lacZ with the lacZ region deleted.
It encodes the first 146 amino acids of B-galactosidase
LacZ is placed into the cloning vector
The other half of lacZ is present in the chromosome.
the strains only express a functional enzyme if they harbour a plasmid that carries the LacZ region.
But if you break open the lacZ to put in a gene of interest it can no longer take part in a-complimentation.

White colonies that form contains an insert, blue colonies contains the plasmid.

Question 23

Where does DNA come from?

Answer 23

PCR or DNA libraries.

Question 24

What is PCR?

Answer 24

Polymerase chain reaction:
Rapid DNA amplification in a plastic tube primer are double-stranded 20 bases long. Can take a few hours.
YOu need to know the DNA sequnce.
Can only be run up to 30 times until all of the components are exhausted.
Use a thermocycler to programme the cycles and temperatures.

Question 25

What are the PCR components?

Answer 25

DNA template Primers to delimit the fragment to be amplified DNA polymerase The 4 nucleotide bases = G,A,C,T

Question 26

What is a library?

Answer 26

collections of DNA fragments from a particular source contained within bacteria or viruses as the host. Can be saved for very long periods and are screened to select out different genes of interest.

Question 27

What types of libraries are there?

Answer 27

Genomic Metagenomic cDNA

Question 28

What is a genomic library?

Answer 28

contains randomly cut peices of DNA isolated from an genome.

Question 29

What is. metagenomic library?

Answer 29

contains randomly cut pieces of DNA isolated from an environmental sample.

Question 30

What are cDNA libraies?

Answer 30

Contains DNA synthesized from RNA using enzyme reverse transcriptase. With no introns.

Question 31

Production of a genomic library

Answer 31

Isolate DNA fragment using restriction enzymes Ligate fragments into plasmids (millions of DNA fragments will be ligated into plasmids)

Question 32

What are the applications of recombinant DNA technology?

Answer 32

Protein hormones Enzymes for washing powders Vaccine epitopes Molecular biology reagents

Question 33

What is DNA replication?

Answer 33

A semi-conservative system Use one template strand to create a complementary DNA strand.

Question 34

How do we synthesis DNA?

Answer 34

Oligo synthesis: Make primers - take a nucleotide and modify it to make binucleotides, trinucleotides, and polynucleotides. DNA synthesis: Primers are 50bp long and overlap 20bp, extension PCr fills in the gaps between the primers, larger DNA molecules can be assembled together using a similar extension PCR process.

Question 35

Advantages of DNA synthesis?

Answer 35

DOenst need a template Can optimise the DNA sequence for a particular organism to help enhance the expression.

Question 36

HOw can we artificially create DNA?

Answer 36

Able to take 1 species remove the DNA take another species and replace the DNA with the first speices' and then it is replicated = synthesis Design of M.mycoides genome, chemical synthesis of oligonucleotides spanning the entire genome, assemble the synthetic intermediate in E.coli, complete the assembly in S.cerevisea, genome transplant to M.capricolum.

Question 37

HOw many genes does an organism need to survive?

Answer 37

MYcoplasma needs 372 genes to survive. Candidatus need 200 genes to survive. The more genes you take away the more blank of a canvas you have to tailor for exactly what you want.

Question 38

What is the minimal genome project?

Answer 38

The minimal amount of genes an organisms needs to survive. Has been synthesised and transplanted into cells with no DNA to form the first artificial organism Mycoplasma laboratorium.

Question 39

How can we alter alreading existing genomes?

Answer 39

Transgenesis and CRISPR

Question 40

What is transgenesis?

Answer 40

Putting an artificial construct into a genome at a random place. Overexpression: Adds new activity or increases the amount of one trait already present. Sense orientation = promoter>ATG>Gene>Stop>Terminator Spider silk in goats Gene inactivation: Represses the expression of a gene present in an organism. Antisense orientation = Promoter>STop>Gene>ATG>Terminator; older technology how "knock out" was discovered. RNAi = Pomoter>ATG> sense gene> antisense gene>ATG>Terminator; doesn't matter which way its done. Repression of PPO in apples

Question 41

What is Cisgenesis?

Answer 41

Only use DNA from that organism.

Question 42

What is CRISPR?

Answer 42

bacterial genes that help protect the bacteria against infection by viruses by recognising and cleaving viral DNA. Manipulated to modify eukaryotic genomes. Gene editing in plants Malaria resistant mosquitos.