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7.012 > Recombinant DNA - Purifying genes > Flashcards

Recombinant DNA - Purifying genes Flashcards

1
Q

To read genes, you firstly need to ____ them.

A

purify (extract from the rest)

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2
Q

Gene

A

particular bit of DNA

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3
Q

Strategy that biochemists already did but that didn’t work with geneticists.

A

purifying proteins by size, charge, binding properties

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4
Q

Gene’s problematic peculiarities

A
  • they are small regions of DNA

- even if a gene is extracted, it will just be an ACTG sequence with no visible properties yet

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5
Q

You can purify genes using:

A

molecular cloning (dilution and amplification)

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6
Q

Strategy to purify genes

A

1) break up DNA into short fragments
2) dilute mix so much that if he were to put a few drops into many different tubes, each tube would get only a single DNA molecule (rather than partitioning based on unique chemical properties)

Partial result: a lot of “pure” yet not useful fractions.
Make useful by: having not one but many DNA copies.
How? Replication

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7
Q

Gene’s perks

A

They can be replicated

Structure of DNA is universal

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8
Q

Molecular cloning’s trick

A

Insert DNA fragments into cells that would work as “tubes”

Great!

  1. they hold the DNA
  2. they replicate DNA fragment during reproduction
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9
Q

Cell based DNA cloning was used since

A

1970s

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10
Q

DNA library

A

large collection of purified DNA fragments,

Note: “Library” can refer to the collection of DNA fragments themselves, the collection of recombinant vectors in which they have been inserted, or the population of host cells that have been transformed with the library.

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11
Q

Search in a DNA library can be done based on:

A

function or protein expression in the host cells

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12
Q

Cloning is…

A

making genetically identical copies of some biological entity.

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13
Q

What can be cloned?

A
  • cells (e.g. mitosis)
  • whole multicellular animals (e.g. Dolly the Sheep)
  • identical twins (occur naturally)
  • molecular cloning (just pieces of dna)
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14
Q

recombinant DNA technology

A

recombining DNA sequences from different sources, to create something not found in nature

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15
Q

What can we do after purifying a gene?

A

We can find the protein it encodes and the molecular-level function that protein performs.

We can do targeted mutations on genes and study their effects on cells, to understand more their function.

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16
Q

Steps for making a DNA library

A
  1. Take some “source” DNA that contains the genes you want to purify, and cut it up into short fragments.
  2. Paste (that is, ligate) the fragments into a vector—some DNA molecule that can direct a cell to replicate it. The result is a library of recombinant vectors.
  3. Deliver the DNA into host cells, a process called transformation. Then physically separate the cells, by plating.
  4. Select cells that have acquired a recombinant vector molecule, and allow them to replicate.
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17
Q

DNA is universal!

A

molecular composition and three-dimensional conformation is essentially the same regardless of whether the DNA came from (human, bacteria, butterfly, elephant, pikachu, bungui, dogui, wilby, buni, jujú)

Also, the enzymes that do the cutting and pasting act on DNA (during molecular cloning) without bias.

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18
Q

enzymes that cut double-stranded DNA at specific sequences

A

restriction enzymes (or restriction endonucleases)

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19
Q

Vector or cloning vector

A

A vector is a DNA molecule that acts as a vehicle for carrying foreign DNA into host cells.

Any piece of DNA that gets replicated in a host cell—separate from the cell’s genome—and that can be used as a tool for carrying DNA fragments into the cell so that they will be replicated.

In many cases, vectors direct host cells not only to replicate them but also to express the genes encoded on them. The most commonly used vectors are engineered versions of natural plasmids.

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20
Q

transformation

A

Uptake of DNA by cells through the cell membrane. (process of delivering the recombinant vectors into host cells)

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21
Q

selectable marker

A

A vector-borne gene that confers a specific trait for which you can perform a genetic selection.

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22
Q

origin of replication

A

a site on the genome where DNA replication begins.

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23
Q

promoter

A

a region of a gene that controls transcription initiation; it recruits RNA polymerase and sets the start site of transcription.

The promoter generally is immediately upstream of the transcription start site (earlier).

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24
Q

DNA ligase

A

an enzyme that can join two pieces of DNA (it catalyses the joining of DNA)

it catalyzes the formation of a new phosphodiester bond between the 3’ hydroxyl group of one DNA strand and the 5’ phosphate of another

The reaction is most efficient when the pieces are already in place by base-pairing H-bonds.

DNA ligase can also catalyze the joining of two pieces of double-stranded DNA that both have blunt ends of any sequence (no sticky overhangs), although that reaction is less efficient.

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25
Q

plasmid

A

A small circular DNA molecule that is separate from a cell’s chromosomal DNA, but that the cell replicates independently from chr. dna.

Plasmids occur naturally in bacteria and many other cells; they are often engineered for use as vectors.

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26
Q

blunt end

A

End of dsDNA in which the two strands are fully base paired.

DNA ligase can ligate two pieces of dsDNA that have blunt ends, but with low efficiency.

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27
Q

sticky end

A

Ragged end of dsDNA—that is, an end at which one strand is slightly longer than the other.

Sticky ends are generally produced by cutting with restriction enzymes.

28
Q

restriction site

A

A specific sequence of double-stranded DNA at which a restriction enzyme binds and makes its cuts.

Most restriction sites are symmetric, meaning that the 5’-to-3’ sequence is identical on the two strands.

29
Q

compatible sticky ends

A

Sticky ends whose overhanging strands have complementary sequences and thus can bind to each other by base pairing.

Two pieces of dsDNA that have compatible sticky ends can be efficiently ligated by DNA ligase.

Two pieces of dsDNA cut with the same restriction enzyme will generally have compatible sticky ends. It’s also possible to generate compatible sticky ends by cutting with two different restriction enzymes that happen to produce complementary overhangs.

30
Q

digest

A

To cut DNA with a restriction enzyme.

An in vitro reaction in which you cut DNA with a restriction enzyme is often called a “restriction digest.”

31
Q

restriction enzymes cut at the same position on both strands (typically because they are ….

A

….homodimers—two copies of the same protein bound together

32
Q

Why do bacteria make restriction enzymes?

A

restriction enzymes serve as a simple defense system that protects bacteria from viruses

33
Q

When a DNA virus infects a bacterial cell,

A

restriction enzymes in the cell chop up the viral DNA, and thus “restrict” the ability of the virus to replicate.

34
Q

Bacteria protect their own DNA by…

A

methylation: Enzymes called methylases add methyl (CH3) groups to specific adenosine or cytidine nucleotides in the genome, which prevents restriction enzymes from cutting it. Viral DNA, lacking these protective tags, is susceptible to cutting by restriction enzymes when it’s injected into the cell.

35
Q

a molecular biologist can order the best restriction enzymes for his/her cloning experiments just by looking at a catalogue nowadays… why?

A

Hundreds of different restriction enzymes have been purified from bacteria already over the last years

36
Q

To take into consideration: restriction enzyme cutting frequency

A

If you want to cut your DNA into many small pieces, you might choose an enzyme whose restriction site will appear frequently (4 basepairs).

if you want longer pieces, you could choose an enzyme with a longer and thus rarer site.

37
Q

The easiest way to generate compatible ends of ligation is by

A

cutting both pieces of DNA with the same restriction enzyme

38
Q

DNA ligase catalysed rxn, more precisely, consists in

A
  1. cutting with enzyme, generating compatible sticky ends
  2. mixing all the pieces and let sticky ends make some complimentary base pairing
  3. Adding DNA ligase to connect the sugar-phosphate backbones
39
Q

compatible sticky ends

A

DNA overhangs (generated when cutting) that are complementary to each other

40
Q

Some restriction enzymes have different restriction sites, but generate:

A

compatible sticky ends

41
Q

You digest this linear DNA molecule with EcoRI. The molecule contains one EcoRI restriction site:

5’ —– GAATTC —– 3’
3’ —– CTTAAG —– 5’

Draw the product(s) of the reaction. Make sure to label the 5’ and 3’ ends.

A

This digestion reaction will produce two products:

5’ —– G 3’
3’ —– CTTAA 5’

and

5’ AATTC —– 3’
​ 3’ G —– 5’

42
Q

You use EcoRI to digest a circular DNA molecule that contains two EcoRI sites. How many DNA products will that reaction produce?

A

two

43
Q

Suppose you digest a circular DNA molecule with the restriction enzyme EcoRI to produce a linear DNA molecule with sticky ends,

Now suppose you want to ligate a piece of DNA (an “insert”) into this linearized molecule to generate a new (and larger) circular molecule. To do that, you first need to trim the insert DNA with a restriction enzyme so that it will have sticky ends that are compatible with the cut ends of your linearized circle. What is the minimum number of compatible restriction sites that the insert needs to have?

A

two

44
Q

Which of these cut inserts would produce a circular molecule when used in a ligation reaction with a molecule that was linearized with EcoRI?

insert 1 (cut with EcoRI):

5’ AATTC ————— G 3’
3’ G ————— CTTAA 5’
insert 2 (cut with SalI):

5’ TCGAG ————— C 3’
3’ C ————— GAGCT 5’
insert 3 (cut with MfeI):

5’ AATTG ————— C 3’
​ 3’ C ————— GTTAA 5’

A

Inserts 1 and 3

45
Q

You digest another circular DNA molecule with the restriction enzyme HaeIII to produce a linear DNA molecule. The HaeIII restriction site is:

​5’ GG/CC 3’
Which of these cut inserts would produce a circular molecule when used in a ligation reaction with the DNA that was linearized with HaeIII?

A) The HaeIII restriction site is:

​5’ GG/CC 3’

B) Eco53kI restriction site:

​5’ GAG/CTC 3’

C) EcoRI restriction site:

​5’ G/AATTC 3’

A

A, B

46
Q

horizontal gene transfer

A

process through which bacteria use plasmids to hand genes back and forth to each other

47
Q

When a bacterial cell dies, what happens?

A

it releases its DNA, including any plasmids it contained.

Another cell can then pick up that DNA and pull it through its cell membrane.

Note: if one of the plasmids carries a gene that makes the cell resistant to an antibiotic compound (produced by other microorganisms around the bacteria), then the recipient cell will get an important competitive advantage.

48
Q

The key sequence features that a plasmid needs to contain in order to serve as a cloning vector are:

A

an origin of replication (ori)
one or more restriction sites
a selectable marker
often, a promoter

49
Q

in a DNA cloning tech, the origin of replication has to

A

recruit the host cell’s DNA polymerase and other DNA replication enzymes to copy the plasmid

50
Q

in DNA cloning tech, restriction sites are so that…

A

the vector can be cut with restriction enzymes so that fragments of your source DNA (foreign DNA “inserts”) can be ligated in

51
Q

in DNA cloning tech, selectable markers allow to

A

select cells that have acquired the vector, and eliminate cells that have not (later in the process)

Note: selectable markers are usually an antibiotic resistance gene

52
Q

in DNA cloning tech, a promoter can

A

drive transcription of any genes in the inserted DNA (see 4.2.3)

53
Q

You plan to make a library of fragments of the yeast genome, ligate them into a vector, and then deliver them into bacterial host cells. You’re looking for a plasmid to use as your vector. In your search, you learn that yeast, like bacteria, contains plasmids that can serve as cloning vectors.

A

a bacterial plasmid (contains a bacterial ori)

Since you plan to transform the recombinant plasmids into bacteria, the plasmids will need a bacterial origin of replication to recruit the bacteria’s DNA replication machinery.

54
Q

Two main things to remember as you make your DNA library:

A
  1. Cut your source DNA and vectors with restriction enzymes that generate compatible ends for ligation (possibilities: same enzyme, diff enzymes with compatible sticky ends, blunt ends)
  2. Use a vector that matches your intended host cells. (origin of replication needs to be recognised by host cells) but not necessarily matching to your source.
55
Q

Suppose you want to make a library of human DNA: then, the source DNA you’ll want to cut up is the ______. If you are planning to use bacteria as your host cells, then as your cloning vector you could use a _________ with _____ ori.

A

human genome, bacterial plasmid , bacterial

56
Q

Why can cut fragments be inserted in any of the 2 orientations into the plasmid?

A

Restriction sites are symmetric, so they generate symmetric sticky ends.

57
Q

Imagine you’ve successfully transformed your recombinant vectors into E. coli. You then realize that you made your library with a plasmid that lacks an origin of replication.

In each of first 3 generations, how many cells do you think will contain the plasmid?

A

1st: 1/2 of the cells
2nd: 1/4 of the cells
3rd: 1/8 of the cells

The genome gets replicated, but the plasmid—because it lacks an origin of replication—does not. So every time a cell divides, the plasmid goes to just one of the two daughter cells.

58
Q

You can deliver your recombinant vectors into host cells, and then select for cells that took up a vector by …

A

plating the cells on selective medium.

59
Q

Why is the efficiency of transformation low?

A

First, many cells won’t take up a plasmidFirst, many cells won’t take up a plasmid

Second, any cells that do take up a plasmid typically get only one.

60
Q

How can you eliminate cells that don’t acquire a plasmid?

A

by performing a selection for cells that have gained antibiotic resistance from a plasmid-borne gene. (ex. adding Ampicillin to test who has Amp R)

61
Q

When do you know that you already accomplished to purify fragments from source DNA?

A

Once you have transformed bacteria with your library, plated the cells, and allowed them to replicate the added DNA.

62
Q

the goal of molecular cloning isn’t just to make many copies of genes, but also to:

A

purify them

63
Q

After transforming the library of recombinant plasmid into host cells, the next step is to

A

spread the mixture across a plate of agar medium, so the cells can grow and divide, and replicate the plasmids

64
Q

Transformation problem: most cells don’t take up a plasmid, and those that do get a plasmid typically get only one.

A

That’s why transformation partitions the plasmids in your library into different cells.

65
Q

What does it mean that replication of plasmids is not always so constrained?

A

the plasmids can be replicated many times per cell cycle. ​

7.012 (9 decks)

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