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Biology 154 > Recombinant DNA technology > Flashcards

Recombinant DNA technology Flashcards

1
Q

What is the central dogma?

A

The flow of DNA to protein, via mRNA

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2
Q

What is replication?

A

using DNA polymerase to make identical DNA molecules

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3
Q

What is transcription?

A

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

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4
Q

What is translation?

A

where proteins are made using mRNA

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5
Q

What is the start codon?

A

Methionine ATG

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6
Q

What is a gene?

A

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

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7
Q

What are regulatory domains?

A

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

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8
Q

How do we know the genes are eukaryotic?

A

Eukaryotic genes have exons and introns
Prokaryotes only have introns

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9
Q

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

A

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

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10
Q

How can we manipulate DNA?

A

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.

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11
Q

What is plasmid?

A

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

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12
Q

What is an antibiotic resistance marker?

A

Identify organisms that contain this plasmid

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13
Q

What is the origin of replication?

A

DNA sequence where plasmid replication starts.

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14
Q

What is the MCS?

A

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.

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15
Q

WHat is an insert?

A

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

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16
Q

What is the promoter region?

A

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.

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17
Q

What is a selectable marker?

A

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

18
Q

What is the primer binding site?

A

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

19
Q

Steps in recombinant DNA tech

A

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.

20
Q

What is DNA-cloning?

A

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.

21
Q

what is a problem of bacterial cloning?

A

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

22
Q

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

A

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.

23
Q

Where does DNA come from?

A

PCR or DNA libraries.

24
Q

What is PCR?

A

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.

25
Q

What are the PCR components?

A

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

26
Q

What is a library?

A

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.

27
Q

What types of libraries are there?

A

Genomic
Metagenomic
cDNA

28
Q

What is a genomic library?

A

contains randomly cut peices of DNA isolated from an genome.

29
Q

What is. metagenomic library?

A

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

30
Q

What are cDNA libraies?

A

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

31
Q

Production of a genomic library

A

Isolate DNA fragment using restriction enzymes
Ligate fragments into plasmids

(millions of DNA fragments will be ligated into plasmids)

32
Q

What are the applications of recombinant DNA technology?

A

Protein hormones
Enzymes for washing powders
Vaccine epitopes
Molecular biology reagents

33
Q

What is DNA replication?

A

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

34
Q

How do we synthesis DNA?

A

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.

35
Q

Advantages of DNA synthesis?

A

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

36
Q

HOw can we artificially create DNA?

A

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.

37
Q

HOw many genes does an organism need to survive?

A

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.

38
Q

What is the minimal genome project?

A

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.

39
Q

How can we alter alreading existing genomes?

A

Transgenesis and CRISPR

40
Q

What is transgenesis?

A

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

41
Q

What is Cisgenesis?

A

Only use DNA from that organism.

42
Q

What is CRISPR?

A

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.

Biology 154 (7 decks)

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