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Alex's Genetics > Lecture 5b > Flashcards

Lecture 5b Flashcards

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Biology 101 flashcards
1
Q

What codes for proteins?

A

The mature mRNA.

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2
Q

How can we copy RNA into DNA?

A

Using an enzyme called ‘Reverse Transcriptase’.

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3
Q

What is Reverse Transcriptase? What commonly has the gene to encode for this?

A

An enzyme that can copy RNA into DNA. Several viruses have the gene that will code for reverse transcriptase.

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4
Q

Does reverse transcriptase need a primer?

A

Yes!

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5
Q

What is cDNA?

A

DNA obtained from RNA through the use of reverse transcriptase.

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6
Q

Describe the process of producing cDNA.

A

1) Anneal a PCR primer.
2) Add reverse transcriptase + dNTPs to synthesize a complementary DNA strand.
3) Denature (separate) the strands.
4) Anneal a second primer
5) Thermostable DNA polymerase with proofreading ability extends, producing a double-stranded cDNA.

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7
Q

What do we need to do after producing the cDNA?

A

The cDNA is then amplified by PCR using the same two primers.

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8
Q

When we go through the process of producing cDNA, what is missing?

A

The promotor of the gene is not produced from this process.

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9
Q

How do we obtain the promotor of the gene?

A

The promotor of the gene is amplified separately by PCR. This generally works because the promotor sequence is short enough to be amplified by PCR.

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10
Q

What is one problem with using Reverse Transcriptase?

A

We do not obtain the promotor from this process.

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11
Q

What is gene cloning?

A

When we combine the promotor and the cDNA.

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12
Q

Where is gene cloning done in?

A

A vector

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13
Q

What do bacterial chromosomes look like?

A

Most bacteria have a single circular chromosome.

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14
Q

Where does bacterial replication of the chromosome occur?

A

For most bacteria, replication of the chromosome initiates at a single site.

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15
Q

What is the origin of DNA replication?

A

This is the location at which chromosome replication begins.

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16
Q

Can DNA polymerase initiate DNA replication on its own?

A

No, it needs an RNA primer to be laid down, and DNA polymerase will then extend from that.

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17
Q

What is the leading strand?

A

A RNA primer is laid down at the origin of replication and DNA polymerase is able to extend throughout the chromosome without the placement of another primer.

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18
Q

What is the lagging strand?

A

RNA primers need to repeatedly be laid down, which results in short bouts of DNA that have to be combined.

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19
Q

What does the replication bubble contain?

A

Leading and lagging strands.

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20
Q

What strand begins at the origin of replication?

A

The leading strand

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21
Q

What strand begins behind the origin of replication?

A

The lagging strands

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22
Q

How many replication forks are used to replicate the entire bacterial chromosome?

A

Two replication forks.

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23
Q

T/F: Three replication forks replicate the entire bacterial chromosome.

A

False, two replication forks replicate the entire bacterial chromosome.

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24
Q

What are plasmids?

A

Small, circular, replicating minichromosomes found in bacteria and yeast. They are separate from chromosomes.

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25
Q

T/F: Plasmids have their own origins of replication.

A

True!

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26
Q

What can occur with some plasmids in bacteria?

A

Some plasmids are able to be transmitted from one bacterium to another.

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27
Q

Do plasmids kill the cells?

A

No, they infect the cell, but they do not lyse the cell.

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28
Q

What do bacteria think of plasmids? What do they do about it?

A

They consider plasmids to be invasive. The CRISPR-Cas9 system acts against plasmids in addition to bacteriophage.

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29
Q

Why can plasmids be good for bacteria?

A

Plasmids can carry genes for antibiotic resistance, which can help the bacteria to survive against antibiotics.

30
Q

What is the best understood bacterial plasmid?

A

The E. coli F factor

31
Q

What does the E. coli F factor do?

A

It encodes for pilli, which promotes contact between bacteria so that they can transfer plasmids through conjugation tubes.

32
Q

How is a plasmid transferred from one bacterial cell to another?

A

Pili will promote cell-to-cell contact so that a conjugation tube can connect the two bacterial cells. The plasmid will replicate and then transfer to the other cell.

33
Q

What is an F- cell?

A

A cell without the F factor.

34
Q

What is an F+ cell?

A

A cell with the F factor.

35
Q

Where do we usually get vectors for gene cloning?

A

Vectors used in gene cloning are usually derived from naturally occurring plasmids.

36
Q

Besides naturally occurring plasmids, where else can we derive vectors from? How does this happen?

A

We can also obtain vectors from viruses. The virus will have sequences removed or mutated so that it can function as a vector without being a pathogen.

37
Q

What are two types of plasmid vectors we can have?

A

High copy number bacterial plasmids and low copy number bacterial plasmids.

38
Q

What is another name for low copy number bacterial plasmids?

A

Bacterial artificial chromosomes (BACs).

39
Q

Describe high copy number bacterial plasmids.

A

They give higher DNA yields when DNA is purified. There is a limit to how much can be inserted into the plasmid.

40
Q

Describe low copy number bacterial plasmids.

A

They can handle having larger DNA fragments inserted into them when a bacterial chromosomal origin of replication is used.

41
Q

What is the AmpR gene?

A

An antibiotic resistance gene that allows bacteria to survive the ampicillin antibiotic.

42
Q

What are restriction enzymes?

A

A defense system against viruses in which proteins made by bacteria will cleave unmethylated DNA sequences.

43
Q

How do bacteria protect themselves from their own restriction enzymes?

A

Many bacteria methylate their own DNA at specific sequences, thus, the restriction enzymes will NOT cleave here.

44
Q

How long are the sequences that get methylated?

A

Usually 4 or 6 base pairs in length.

45
Q

Who uses restriction enzymes? Why?

A

Bacteria and plasmids use restriction enzymes as a defense system against viruses.

46
Q

What do restriction enzymes do when a virus infects?

A

The viruses that infect the bacteria have unmethylated DNA, thus, the restriction enzymes cleave it.

47
Q

Describe an overview of how bacteria use restriction enzymes.

A

Bacteria protect their own DNA by methylating it and then letting restriction enzymes cleave unmethylated DNA that viruses carry in.

48
Q

Where do restriction enzymes cleave DNA?

A

They bind to DNA sequences and then cleave the DNA at two defined locations, one on each strand.

49
Q

What types of ends can restriction enzymes produce?

A

Sticky/Staggered ends and Blunt ends.

50
Q

Why types of sequences are restriction enzymes cleaving?

A

Palindromic sequences, which are sequences that read the same forward one on strand as they read backwards on the other strand.

51
Q

What are sticky/staggered ends? What does the shape of the cut look like?

A

After the restriction enzymes cleave the strands, they are still able to hydrogen bond. Shapes of the cuts appear as L’s.

52
Q

What are blunt ends?

A

The restriction enzyme cleaves the strands right down the middle, so there are no single-stranded ends sticking out.

53
Q

When we are cloning genes, what type of ends do we want to produce with restriction enzymes?

A

We want to produce sticky ends so that we can put pieces together.

54
Q

How can we combine DNA from 2 different sources to produce a recombinant DNA molecule?

A

1) We take DNA from 2 different sources and incubate them with EcoRI, which cuts the DNA into sticky ends.
2) Incubate the DNAs together, allowing sticky ends to hydrogen bond.
3) Add DNA ligase, which will covalently link the DNA backbones.

55
Q

For gene cloning, how do we obtain the promotor?

A

The promotor is amplified using primers that have restriction enzyme cleavage sites at the 5’ ends.

56
Q

What is special about the primers used to amplify the promotor in gene cloning?

A

There is an extra sequence attached to the 5’ end of the primer that acts as a site for restriction enzyme cleaving to produce sticky ends.

57
Q

In regards to the promotor, what does cleavage with restriction enzymes do?

A

The restriction enzyme cleavage gives the promotor sticky ends.

58
Q

Once we have the cDNA, what do we do?

A

We have to PCR amplify it using primers with restriction enzyme cleavage sites. The restriction enzymes cleave the primers and we get sticky ends.

59
Q

On the sticky ends, what is special about the 3’ and 5’ ends?

A

On one side of the promotor or cDNA, the 3’ end has the sticky end. On the other side, the 5’ end has the sticky end. It looks like a Z.

60
Q

How do we insert a PCR product into the plasmid vector?

A

The PCR product and plasmid vector are cleaved with the same restriction enzyme so that their sticky ends match. DNA ligase then covalently links the ends.

61
Q

Once we have inserted the promotor, what do we need to do?

A

We just have the promotor in there right now so we need to clarify that it made it into the plasmid. To do so, we take the plasmid and put it back into the bacteria.

62
Q

What gene should the inserted promotor be disrupting?

A

The LacZ gene.

63
Q

What does the LacZ gene do?

A

It encodes a protein that cleaves X-gal.

64
Q

What does intact X-gal appear as?

A

Colorless.

65
Q

What does cleaved X-gal appear as?

A

Blue

66
Q

How do we know if our inserted promotor disrupted the LacZ gene?

A

Colonies produced by the bacteria will appear colorless because we disrupted the function of the LacZ gene by inserting the promotor there.

67
Q

What color colony would we want to then perform a PCR with?

A

White colonies because these are the colonies where the promotor was successfully inserted.

67
Q

T/F: Each bacterial colony is derived from a single cell. Thus, all the cells in a colony are genetically identical.

A

True!

68
Q

In regards to the plasmid, what is important for picking a colony we will insert the cDNA in?

A

We need to make sure we obtain a plasmid with correct orientation before inserting the cDNA.

69
Q

What do we not want to do when we insert the cDNA?

A

We do not need to use the LacZ approach this time because we already know the promotor disrupted it.

70
Q

After we have inserted the cDNA and promotor into the bacterial vector, what do we now do to get the trans gene?

A

We excise the transgene from the plasmid and insert it into the ROSA26 locus using CRISPR-Cas9.

Alex's Genetics (38 decks)

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