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Molecular Biology > Ch. 7 Studying Genes > Flashcards

Ch. 7 Studying Genes Flashcards

1
Q

What is a genome?

A

A genome is ALL the genetic information of a cell, which codes for all functions. (46 chromosomes, mito DNA, plasmids in bacteria…)

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2
Q

What are the biotechnologies and how are they related to studying genes?

A

The techniques used to study genes are innumerable, but this section focuses on those related to biotechnologies: medicinal, agricultural, manufacturing use, etc.

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3
Q

What is gene cloning?

A

The replication of a gene from one species into another species.

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4
Q

What is a gene of interest?

A

A gene that is targeted and acquired, then inserted into a cloning vector using DNA ligase.

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5
Q

What does the insertion of a GOI into a cloning vector produce?

A

This produces recombinant DNA: DNA from two different sources that are combined into one piece.

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6
Q

How is a GOI actually cloned?

A

The recombinant vector (GOI and cloning vector) are placed into cells, and cells that have acquired the vector are selected for.

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7
Q

What are the three types of cloning vectors?

A

Plasmids, bacterial artificial chromosomes, and yeast artificial chromosomes.

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8
Q

When are plasmids used as a cloning vector?

A

Plasmids can be used for cloning up to ~10,000 bp.

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9
Q

What are bacterial artificial chromosomes and when are they used as a cloning vector?

A

They are specialized plasmids that can receive up to ~300,000 bp.

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10
Q

What are yeast artificial chromosomes and when are they used as a cloning vector?

A

They are plasmids modified to replicate in eukaryotes and can carry up to ~2,000,000 bp.

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11
Q

What are DNA libraries?

A

Collections of multiple large fragments of DNA that have been cloned.

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12
Q

How are DNA libraries created?

A
  • whole genomes are cleaved into thousands of pieces
  • each piece is cloned
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13
Q

Are DNA libraries required in today’s research?

A

No. Newer sequencing technologies DON’T require libraries, but early genome sequencing did require them.

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14
Q

What is cDNA?

A

complementary DNA; DNA complementary to cellular RNA

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15
Q

How can a cDNA library be made (4)?

A

It is made based on the mRNA present in a cell.
-mRNA is collected from cells (treat with DNase)
- reverse transcriptase is used to make a DNA copy of the transcripts
- mRNA is degraded and PCR is used to make a compliment to the cDNA
- the ds fragments are cloned into vectors

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16
Q

How can a primer for cDNA PCR be made?

A

Eukaryotic RNA has a poly-A tail, so and oligo-dT primer can be added to the sample.

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17
Q

What is PCR?

A

Polymerase chain reaction allows for the SPECIFIC amplification of desired portions of a genome.

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18
Q

What does conventional PCR require?

A

It requires DNApol, DNA primers that DNApol extends, and a target DNA sequence.

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19
Q

What is the “bare bones” process of PCR?

A
  • the primers anneal to complementary sites in the DNA and determine what portion of the DNA is amplified

-template DNA is amplified by DNApol in vitro (in a tube)

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20
Q

Why is PCR used in biotechnology, research, diagnostics, and more?

A

It is used because of its high sensitivity!

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21
Q

What is RT-PCR?

A

Reverse transcription PCR; it uses an RNA template to make DNA. This is how a cDNA library is made.

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22
Q

What is quantitative PCR?

A

Also referred to as qPCR or real-time PCR; it records the amount of DNA present after each cycle to quantify the amount of DNA present in the original sample.

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23
Q

Does conventional PCR give you an amount of DNA?

A

No. It only tells you presence or absence of DNA.

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24
Q

If you combine qPCR and RT-PCR what can you determine?

A

You can determine the amount of a specific transcript (RNA) present in a sample.

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25
What is Sanger Sequencing?
A sequencing method that is conceptually similar to PCR.
26
How is Sanger Sequencing done?
- primer binds the template DNA to be sequenced - pol adds complementary nucleotides (dNTPs) - some nucleotides are ddNTPs that cause chain termination - produces many strands of DNA all differing in length and size
27
How was Sanger Sequencing initially performed, and how is it done now?
It was originally done with radiolabeled ddNTPs, but those are bad for health. Now it is done with fluorescent labeled ddNTPs.
28
What is Sanger good for?
Sanger is good for sequencing small pieces of DNA like single genes, operons, plasmids, etc.
29
How many bases can Sanger sequence per reaction?
~600-900 bases per reaction.
30
Can Sanger sequence a whole genome?
It can, but it is very slow and expensive for that amount of DNA.
31
What are the new sequencing techniques know as?
They are called next generation sequencing (next gen.) and have some similarities to Sanger, but some big differences too!
32
What is pyrosequencing?
Pyrosequencing, also known as 454 sequencing, is a next gen. technique.
33
What is the process of pyrosequencing/454 sequencing (4)?
- DNA is sheared - an oligonucleotide tag is ligated to all fragments, providing a known starting point for all pieces - all fragments are immobilized & each is amplified by PCR creating a cluster of each fragment on a surface - all the fragments are sequenced at the same time
34
What is the chemistry of pyrosequencing/454 sequencing (5)?
- individual dNTPs are pulsed (added) - any unused dNTP is degraded by apyrase - if the pulsed dNTP is added, pyrophosphate (PPᵢ) is released and converted to ATP by sulfurylalse - ATP causes luciferase to produce a flash of light - a detector records where flashes of light occur at each location on the surface of the chip
35
What does a brighter flash during 454 sequencing indicate?
A brighter flash means that more than one of the base that was pulsed was added.
36
What is reverse terminator sequencing/Illumina?
Illumina is a next gen. sequencing technique similar to 454 sequencing, but it uses fluorescently-labeled dNTPs instead.
37
What do the Illumina labeled dNTPs have on them that the 454 dNTPs don't?
They have a blocking group that prevents the addition of another nucleotide, so only one nucleotide can be added at a time.
37
What is the process of Illumina sequencing (6)?
- a pulse of all four labeled dNTPs is added - one dNTP is added to each growing strand - a laser excites the fluorescent probe - an image of the surface is taken to record the color added - a chemical removes the blocking group for the next dNTP - process repeats
38
How many bases can next gen. techniques sequence?
They can reliably sequence 250-500 bp fragments, but they can sequence thousands of fragments at a time. (Sanger can do 1000 bp, but only one fragment at a time).
39
Why is next gen. not done for simple sequencing?
Running one chip can be VERY expensive, so it is too expensive for simple sequencing.
40
What is next gen. good for?
It is great for large sequencing projects like whole genomes.
41
How do you assemble a whole genome from 500 bp pieces?
Computers! Computers find overlapping parts of fragments (contigs) and assembles them based on overlap.
42
What does accuracy of sequencing rely on?
It relies on sequencing depth which is the number of times a single base position is sequenced in a fragment. The more the better!
43
How are cloned genes expressed?
They are expressed from expression vectors (i.e. pBAD) that contain promoters recognized by the host cell.
44
What do expression vectors utilize to make sure only transformed cells are present?
They utilize inducible promoters to make sure expression is controlled (i.e. ara).
45
What are affinity tags?
Chemical tags that can be added to the end of a protein for purification or immunoassays (i.e. 6X His tag).
46
What is site-directed mutagenesis?
Mutagenesis that is used to modify specific amino acids of a protein
47
What can site-directed mutagenesis be used for (3)?
It can be used to determine the function of an amino acid, domain, or the whole protein.
48
How can mutagenesis be accomplished (4)?
restriction enzymes PCR zinc finger nucleases (in vivo: in the cell) CRISPR/Cas (in vivo: in the cell)
49
How do restriction enzymes carry out mutagenesis?
Restriction enzymes can be used to cut out a portion of a coding sequence.
50
How can PCR carry out mutagenesis (2)?
PCR primers can carry a mutation, so amplicons are mutated. PCR products can also be designed to introduce additions or deletions to a specific gene.
51
How do zinc finger nucleases carry out mutagenesis?
They are "designer" nucleases created to target a specific DNA sequence. - create a dsDNA break - the breaks can be repaired, but nucleotides are lost before the repair is made, leading to mutations in the target gene
52
How does CRISPR/Cas carry out mutagenesis?
It is a newer technology that allows for targeted mutation of prokaryotic or eukaryotic genes.
53
Where does CRISPR/Cas naturally occur?
It is naturally occurring in bacteria as a phage defense mechanism.
54
How can natural CRISPR/Cas be modified and what does this do (2)?
It can be modified to target specific genes for mutation by designing target RNA. It can also insert DNA using homologous recombination to introduce new genetic information to the cell.
55
How can gene and protein function be studied (5)?
Protein fusion Western blotting Yeast two-hybrid assays Coimmunoprecipitation Microarrays
56
What is protein fusion?
When a reporter gene, like GFP, is added to a gene being studied.
57
What can protein fusion be used to study (3)?
It can be used to study protein localization, expression during development, and regulation (for regulation studies link the reporter gene to a promoter of interest).
58
What is western blotting?
It is used to detect the presence of a protein in a cellular extract.
59
How are protein-protein interactions detected (2)?
Protein-protein interactions can be detected using yeast two-hybrid assays or coimmiunoprecepitation. (Coimmunoprecipitation works because there is so much antibody it literally precipitates out of solution).
60
How is gene expression of a cell measured?
The gene expression of a cell (all the transcripts) can be measured using microarrays.

Molecular Biology (11 decks)

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