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Alex's Genetics > Lecture 4b > Flashcards

Lecture 4b Flashcards

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Biology 101 flashcards
1
Q

What is the easiest way to delete or alter a gene?

A

CRISPR-Cas9 System

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2
Q

What are operons?

A

In bacteria (but not eukaryotes), clusters of genes that produce a RNA that serves as the mRNA for all the genes in the cluster.

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3
Q

What is the product of the operon?

A

A long RNA called Polycistronic mRNA.

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4
Q

How are genes in an operon related?

A

While they each code for different proteins, the genes within each group are always dedicated to the same biochemical pathway. In other words, they are all going to work towards the same physiological effect.

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5
Q

What precedes the coding sequence in the polycistronic mRNA?

A

Shine-Delgrano Sequences, which are a sequence that causes a ribosome to load.

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6
Q

Is Eukaryotic mRNA polycistronic?

A

No

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7
Q

Does Eukaryotic mRNA have Shine-Delgrano sequences?

A

No. Their mRNA is not polycistronic.

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8
Q

Where does the CRISPR-Cas system come from?

A

It comes from bacteria, because bacteria use this system as a defense against bacteriophages (viruses that infect bacteria).

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9
Q

How many genes are in the CRISPR-Cas operon? What are they?

A

5 genes: tracr, Cas9, Cas1, Cas2, and Crispr.

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10
Q

Where does the name CRISPR come from?

A

The Crispr gene has a group of Clustered Regularly Interspaced Palindromic Repeats.

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11
Q

What are the repeats?

A

pre-crRNAs, a non-coding type of RNA.

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12
Q

What are the repeats spaced out with?

A

The repeats are interspersed with short, unique sequences. They are always the same length, however, each spacer has a different DNA sequence.

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13
Q

What do we mean when we say that the CRISPR-Cas system is an Adaptive Defense System?

A

This means that a bacterial cell must first be exposed to an agent (bacteriophage) before it can elicit a response.

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14
Q

How many phases does CRISPR-Cas defense system occur in? Name them.

A

The defense mechanism occurs in 3 phases:

1) Adaptation
2) Expression
3) Interference

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15
Q

What is another term for the adaptation phase?

A

The spacer acquisition

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16
Q

Describe the steps in the Adaptation Phase.

A

1) A bacteriophage infects a bacterium.
2) Cas1 and Cas2 genes are expressed to create proteins that form a complex. This complex will cleave the invading bacteriophage DNA into tiny parts.
3) The pieces of this bacteriophage DNA can be inserted into the Crispr gene.

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17
Q

Where do the spacers in the Crispr gene come from?

A

The cleaved bacteriophage DNA from past infections.

18
Q

T/F: The spacers in the Crispr gene can be passed to daughter cells.

A

True. This means that any replicated cell of the original bacterial cell will have this defense system from previous infections.

19
Q

What genes get expressed in the expression phase?

A

The Crispr, tracr, and Cas9 genes.

20
Q

What genes get expressed in the adaptation phase?

A

The Cas1 and Cas2 genes.

21
Q

What are two non-coding RNAs?

A

pre-crRNA and tracrRNA

22
Q

What is the pre-crRNA?

A

These are the palindromic repeats in the Crispr gene. They are interspersed between the spacers.

23
Q

Describe the steps of the Expression Phase.

A

1) The genes that encode for pre-crRNA and tracrRNA are transcribed.
2) Several tracrRNAs will base-pair with the pre-crRNA due to complementary base.
3) The pre-crRNA is cleaved into several crRNAs, now called crRNAs.
4) The tracrRNA will form a complex with crRNA and this complex binds to the Cas9 protein from a recognition site in the tracrRNa.

24
Q

Each spacer in a crRNA is complementary to…

A

One strand of the bacteriophage DNA. This means that the crRNA serves as a GPS to find the bacteriophage DNA.

25
Q

Describe the steps in the Interference Phase.

A

1) The crRNA will lead the tracrRNA-crRNA-Cas9 complex to bind to the bacteriophage DNA strand.
2) The Cas9 protein acts as an endonuclease to make double-stranded breaks in the bacteriophage DNA. This inhibits phage proliferation.

26
Q

When Crispr-Cas9 is used to edit the genome, what acts as the ‘Guide RNA’?

A

The crRNA and the tracrRNA are one big Guide RNA.

27
Q

When using Crispr-Cas9 to edit the genome, what is the role of the Cas9 protein?

A

It cleaves both strands next to the genomic target sequence.

28
Q

Once the Cas9 protein cleaves the strands, what happens?

A

The cell will use Non-Homologous End Joining (NHEJ) to rejoin chromosomes.

29
Q

What method can we use to determine that Crispr-Cas9 actually altered/deleted a gene?

A

The easiest way uses the Polymerase Chain Reaction (PCR).

30
Q

How can we separate DNA strands?

A

If we heat DNA to 94 degrees Celsius, the strands will separate.

31
Q

After we separate the strands in PCR, what do we do?

A

We mix in several copies of a single-stranded DNA fragment and let the DNA cool.

32
Q

How do the smaller DNA fragments outcompete the longer DNA?

A

Because there are so many more copies of the short DNA fragments.

33
Q

What is primer extension?

A

Using a chromosomal DNA fragment as a template, DNA polymerase will add nucleotides to the 3’ end.

34
Q

What is an oligonucleotide?

A

A short single-stranded fragment of DNA that can latch onto a single strand of DNA.

35
Q

What is a primer?

A

When an oligonucleotide is used to synthesize DNA.

In other words, this is when a short single-stranded fragment of DNA latches on the a single strand of DNA and is then extended by DNA polymerase.

36
Q

What is Taq?

A

A heat-resistant DNA polymerase that is isolated from a bacteria that can withstand high temperatures.

37
Q

Why do we need Taq?

A

Heat destroys normal polymerase, and we need to do many cycles of PCR.

38
Q

What the steps of PCR?

A

1) Heat/Melt
2) Anneal
3) Extend

39
Q

T/F: Primers can be too far apart for PCR to work.

A

True.

40
Q

How does using PCR allow us to see if Crispr-Cas successfully deleted a gene?

A

PCR products allow us to obtain a DNA sequence that can be matched to the known DNA sequence from our gene.

Alex's Genetics (38 decks)

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