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Y1 Genes, Molecules and Cells S2 > Genetic Engineering > Flashcards

Genetic Engineering Flashcards

1
Q

What is genetic engineering

A

Directly manipulating the genetic material of a phage, cell or whole organism for a specific purpose

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2
Q

Genetic engineering requires what type of technology

A

Recombinant DNA technology
(both in vitro and vivo)

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3
Q

Name 4 basic features of DNA

A
  • Double stranded
  • Antiparallel
  • Watson Crick base paring held together by H-bonds
  • From 5’ to 3’ bonding
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4
Q

How can DNA be extended

A

A DNA strand can only be extended in one direction - extended by adding nucleotides to the 3’ hydroxyl end

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5
Q

What is gene cloning

A

Propagation of multiple identical copies of DNA for study/manipulation etc

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6
Q

What enzyme is needed to extract DNA from a cell
Why is it useful?

A

Restriction enzymes (restriction endonucleases)
Allows target of a specific recongition sequence

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7
Q

Once we have the acquired section of DNA, what happens then?

A
  • A plasmid is cut with the same restriction enzyme and gene is inserted using DNA ligase
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8
Q

Once we have the plasmid with inseted DNA, what do we then do

A
  • Transform recombinant plasmid into a bacterial host and amplify the clone
  • Then isolate plasmid DNA from a large number of bacterial cells
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9
Q

What is the difference between a restriction Endonucleases and exonucleases

A

Endonucleases cut DNA in the middle
Exonucleases that attack nucleic acids from the end

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10
Q

How can bacteria protect its own DNA (such as from a phage)

A

Restriction/modification system
The bacteria chemically modifies these specific sequences (by methylation) which prevents cutting
leads to forgein DNA being cut up and destroyed
Modification enzymes works as a protector from the bacterial DNA from being cut up

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11
Q

Which organism do restriction enzymes originally come from

A

Bacteria
(now made from isolated genes on a commercial level)

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12
Q

How do Type 1 restriction endonucleases operate

A

These enzymes recognise their target sequence then cut the DNA at a random position far from this sequence

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13
Q

How do Type II restiction endonucleases operate (most common type of endonucleases)

A

Type II enzymes usually cut within the recognition site

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14
Q

How big are these recognition sequences usually

A

Either 4, 6 or 8 base pair sequences
4, 6, or 8 cutter)
work as dimers

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15
Q

How do type III enzymes operate

A

These enzymes cut a short distance away from the recognition site and need two recognition sites in opposite oritentations in the same piece of DNA

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16
Q

How do type IV enzymes operate

A

Type IV enzymes only cut DNA with specific chemical modifications (e.g. methylated DNA)

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17
Q

Type II enzymes usually cut at what type of sequence

A

Palindromic sequence: word spelt same way backwards as forwards

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18
Q

How do Type II restiction enzymes cut at palindromic sequences

A

They work as dimers - two subunits of the enzyme that work together and each monomer cuts one of the two strands

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19
Q

If some type II restriction enzymes were to cut asymmetrically vs symmetrically, what is produced

A

Asymmetrically: overhanging single-stranded sticky ends
Symmetrically: producing blunt ends

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20
Q

Why is producing sticky ends more useful

A

Sticky ends are useful for cloning as DNA cut with the same enzyme can be easily stuck together as the sticky ends will readily base pair with each other
This can either be a 5’ or a 3’ overhang

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21
Q

Is it the smaller or larger plasmids which are used for cloning

A

Smaller
Easier to manage and use

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22
Q

What is the benefit of plasmids

A

Plasmids carry extra genes that may be required by the cell in specific circumstances - overall giving an advantage
e.g. to confer antibiotic resistance, nitrogen fixation, amino acid biosynthesis etc

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23
Q

Only some bacterial cells within a population may possess a particular plasmid at any given time
How can horizontal evolution of this plasmid occur

A

Some plasmids can be copied from one bacterial cell to another by bacterial ‘sex’ (conjugation) allowing horizontal evolution
(keep in mind only large plasmids can conjugate not small ones)

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24
Q

Here is the basic structure of a large plasmid
What is the function of tra operon

A

tra operon = transfer gene (needed for conjugation)
It determines the synthesis of a structure called a sex pilus
This brings two cells together and triggers plasmid transfer

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25
Q

Here is the structure of a large plasmid
What is the difference between OriV and OriT

A

OriV - plasmid origin of DNA replication
OriT = origin of transfer

26
Q

Describe the process of conjugation, where a whole plasmid is transferred

A
  • The sex pilus makes contact and draws the cells together to form a conjugation bridge from the F+ to F- bacteria
  • Double stranded plasmid DNA is nicked (one strand is broken) at oriT and rolling circle DNA replication initates from nicked 3’ end
  • The displaced strand is directed into the F- cell (5’ end first) where it gains a complementary strand
  • Cells the seperate
27
Q

What is the formation of a Hfr strain

A

Lead to transfer of some of the chromosomal DNA from one cell to another

28
Q

What site allows the formation of the Hfr strain

A

the IS elements
(insertion sequence)

29
Q

When smaller plasmids are used as vectors, what two things are added to them

A
  • Usually engineered to carry added antibiotic resistance (selectable marker) genes
  • Also engineered to carry an added multiple cloning site (site with losts of restriction enzyme cut sites to allow you to easily insert DNA)
  • Stuck using DNA ligase
30
Q

Where would the restriction sites likely be found on a small chromosome

A

Within the antibiotic resistance genes
This allows the resistance genes to be ‘insertionally inactivated’ which is useful in clone identification, due to the gene to cease functioning

31
Q

E. coil cells are treated with what so they take up the plasmid DNA

A

Calcium chloride

32
Q

In modern plasmids for genetic engineering, there is usually one antibiotic resistance gene and a lacZ’ gene
Why is the lacZ’ gene added

A

Foreign DNA inserted into the multiple cloning site destroys the activity of the lacZ’ gene (no longer produces functional protein)
Complete lacZ gene encodes β-galactosidase which cleaves X-gal within agar, resulting in insoluable blue dye = blue colonies
the recombinant colonies will be white

33
Q

Recombinant plasmids need to be inserted into E.coli using transformation
E.coli is not naturally competent so won’t take up plasmid naturally
What are the 3 methods to do this?

A

1) CaCl₂ precipitation and heat shock (used in bacteria)
2) Electroporation (used in bacteria)
3) Gene gun (used in plant genetics)

34
Q

Once we have identified a recombinant colony on the agar plate (white), what would we then do?

A
  • Pick a single colony and grow bacteria in appropriate growth media and conditions overnight
  • Lyse bacteria and isolate plasmid DNA
35
Q

When we are isolating DNA, bacteria are treated with the detergent sodium dodecyl sulphate (SDS) in the presence of sodium hydroxide to ensure an alkaline pH
What does the SDS do

A
  • SDS disrupts the cell membrane and denatures proteins as well as the chromosomal E.coli DNA which has a lot of proteins associated
  • The plasmid DNA remains in solution
  • Neutralisation results in aggregation of chromosomal DNA, protein-SDS complexes and heavy RNA
36
Q

How is the aggregated DNA in the SDS solution isolated and removed

A

The precipitate is removed by centrifugation and the plasmid DNA remains in solution
A silica exchange column is then used which the plasmid DNA will bind to at high salt concentration and contaminants can be washed away

37
Q

Pure plasmid DNA can then be separated on a …… according to determine the conformation state/quality of extract

A

Agarose gel eletrophoresis

38
Q

How does argarose gel electrophoresis work

A

DNA is negatively charged
DNA fragments separated on the basis of their size by applying an electrical current
The bigger the fragments, the less it moves down the gel

39
Q

DNA must be stained to make it visible
What is the most common technique to do this

A

Ethidium bromide which is added to DNA before electrophoresis is run
Which is fluorescent under UV

40
Q

How can we determine the length of DNA fragment of a unknown size

A

Using a size marker
A size marker is a set of DNA fragments of known sizes
At the end of electrophoresis this distance migrated down the gel is recored and using the plot of distance migrated against Log₁₀ fragment size, the size of the unknown fragment can be worked out

41
Q

From the agarose gel, DNA can be transferred
One technique is Southern blotting, what does this involve?

A
  • DNA is seperated through an agarose gel and blotted onto a nitro-cellulose membrane (drawn up through a buffer solution)
  • We can soke the membrane in a solution containing a radioactively labelled DNA probe which will hybridise with a specific DNA sequence in one of the fragments
  • And expose to x-ray to work out the location of this fragment
42
Q

What are the other two blotting techniques based on Southern blotting

A

Northern Blotting - transfer of RNA from gel to membrane
Western Blotting - transfer of proteins from gel to membrane

43
Q

What is cDNA?

A

Double stranded DNA made by copying eukaryotic mRNA

44
Q

How is cDNA formed

A

the mRNA is copied into single stranded DNA using a reverse transcriptase enzyme (from retrovirus)
And then this single stranded DNA is made double stranded using DNA polymerase

45
Q

What is the benefit of cDNA

A
  • all the introns have been removed - bacteria cannot remove introns
  • Easily inserted into a plasmid vector and used to make protein product of the gene in prokaryotes/eukaryotes
46
Q

How is cDNA made (on a molecular level)

A
  • mRNA at the 3’ end has a poly(A) tail, which an added Oligo(dT) primer is added to
  • Use reverse transcriptase to synthesis a single strand of DNA
  • the cDNA forms this hairpin loop, forming another 3’ end
47
Q

What is the benefit of cDNA forming this 3’ hair pin loop

A

On adding DNA polymerase and bases, the DNA can continue extending from the 3’ hairpin loop
Then once two strands are formed, endonuclease can cut the loop to form two individual strands

48
Q

What is the use of the polymerase chain reaction (PCR)

A

PCR is the amplification of a specific DNA sequence in vitro (usually in a small, thin walled, snap cap tube)

49
Q

What are two large benefits of PRC

A
  • DNA can be aplified from a very low level to a level that can be seen easily by agarose
  • DNA can be specifically amplified from a sample containing a whole mix of different DNA sequences (doesn’t need to be pure/clean)
50
Q

What are the 5 things required for PCR

A
  • Sample of DNA
  • Primers
  • Heat stable DNA polymerase
  • Buffer
  • building blocks of DNA (dGTP, dATP, dTTP known as dNTPS)

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51
Q

What are primers

A

short, single stranded oligonucleotides, each complementary to a different strand of the required DNA fragment
Primer 1: forward primer has the same sequence as the top strand of DNA
Primer 2: reverse primer and has the same sequence as the bottom strand of the DNA

52
Q

What is the basic method of PCR

A

Repetitive heating (denature), cooling (annealing) and DNA synthesis (extension) amplify the DNA between the primer binding sites to yield large quantities of DNA

53
Q

5’-3’ polymerase and 3’-5’ exonuclease work as a pair in E. coli, how?

A

3’-5’ exonuclease proofreads the nucleotide that the 5’-3’ polymerase has just added, and potentially remove it, if the wrong one
(note these enzymes cannot work in PCR due to high temps)

54
Q

What is the name of the thermostable DNA polymerase used in PCR

A

Taq polymerase
But lacks a 3’-5’ exonuclease proofreading activity

55
Q

On heating the DNA to 94 degrees in PCR, the two strands will seperate
Then it is cooled to 50-65 degrees, why?

A

Allows the primers to base pair with their complementary strands (annealing)

56
Q

Once cooled to 50-65 degrees, the sample is then reheated to 72 degrees, why?

A

Allow the Taq polymerase to synthesise two new DNA strands by adding nucleotides to the forward and reverse primers (extension)

57
Q

One PCR sample ……. the amount of the DNA fragments

A

Doubles

58
Q

When using two primers in PCR, what feature of the two primers should be similar

A

Annealing temperature of the two primers should be approximately equal

59
Q

Why would we try to avoid complementary sequences within a primer

A

To prevent the formation of a hairpin loop, as well as between the two primers

60
Q

When PCR has replicated DNA, the ends of the copies are not blunt (an extra A is added to 3’ end of each strand), causing an issue for inserting DNA into plasmid
How is this overcome

A

Adding recognition sites for restriction enzymes at the ends of the primers

61
Q

What is RT-PCR?

A

reverse transcriptase-PCR
Needed to amplify mRNA sequences, through converting to DNA using reverse transcriptase, then carrying out PCR

62
Q

What does Quantitative PCR allow

A

Allows the synthesis of a PCR product to be followed over time (mainly monitored in early stages while reaction is still linear)
Important in precise quantitation of the amount of starting template

Y1 Genes, Molecules and Cells S2 (12 decks)

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