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BIOL226 > Topic 7 > Flashcards

Topic 7 Flashcards

1
Q

Which of the following repair mechanisms does not make use of a polymerase?

Direct repair
Nuclear excision repair
Mismatch repair
Non-homologous end joining
None of the above

A

Direct repair

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2
Q

What are the recombinant DNA techniques used to manipulate DNA?

A

Cloning: synthesize (polymerize) thousands of DNA copies
PCR: detect DNA fragments
Gel electrophoresis: quantify DNA fragments
Southern blot: retrieve (purify) DNA fragments

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3
Q

Define gene cloning

A

Introducing a desired DNA molecule that can be introduced and propagated in vivo inside a host cell, then extracted and purified

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4
Q

Define a recombinant DNA molecule

A

two or more different DNA molecules joined together

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5
Q

Summarize the process of cloning

A

(1) Insert purified DNA isolated via restriction digestion or PCR
(2) Plasmid (vector) cut open with a restriction digestion and insert digested with restriction enzymes to make matching ends, then ligase joins insert and plasmid backbone
(3) Transform ‘competent’ bacteria with ligation products

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6
Q

Describe the cloning rationale.

A
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7
Q

Which is true about reverse transcriptase?
Only found in bacteria
transcribes RNA into DNA
is not used in gene cloning
only found in the nucleus
none of the above

A

Always transcribes RNA into DNA

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8
Q

Describe reverse transcription (copying genes from mRNA).

A

Reverse transcription converts mRNA to cDNA (complementary DNA: double stranded DNA copy of an RNA), because it is easier to isolate genes of interest from mRNA than the genome. However, part of the sequence to ‘prime’ reaction must be known.

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9
Q

Describe polymerase chain reaction (PCR), an in vitro system to amplify DNA fragments.

A

‘Making’ DNA, or excising and amplifying DNA between two primers for purification.
Note that the primers require a known DNA sequence.

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10
Q

What is required for a PCR reaction?

A

Template DNA- from cells
Primers- synthesized in vitro
dNTPs- to make more DNA
Buffer- to make ideal environment for enzymes
DNA polymerase- to synthesize DNA
- must be resistant to high temperature
- Taq polymerase is a hotspring bacteria

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11
Q

How is DNA purified?

A

Agarose gel electrophoresis separates DNA fragments
- DNA is negatively charged: migrates towards the positive pole
- DNA fragments will separate from each other based on size (bp)
- Long DNA fragments migrate slower through agarose matrix

DNA is observed in gels using a fluorescent DNA intercalator (Ethidium Bromide, etc.)
DNA can be extracted and purified from bands on agarose gels

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12
Q

Define plasmid.

A

a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes.

Note that many carry antibiotic resistance genes, which can be used as selectable markers.

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13
Q

What are vectors?

A

Vectors are small, transferrable, and replicable DNA molecules that must be recognized and replicated in a host, such as plasmids (bacteria) or phage DNA (virus)

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14
Q

What is a plasmid?
bacterial DNA
passed between bacteria
double stranded
circular
all of the above

A

All of the above

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15
Q

What are the essential assets of a functional plasmid re-engineered for cloning?

A
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16
Q

Describe the process of cloning with λ phage.

A

Capacity for genome packaging into virus is 45Kb
The 15Kb central portion of the λ-phage genome with lysogenic genes, dispensible for replication and packaging, is removed for cloning.
That leaves a possible insert of up to 15Kb
Inserts assembled in vitro as concatemers (series of genomes linked by COS sites) before being spliced into 45Kb single genomes and packaged.
Packaged phage ready for infecting live bacterial host for propagation.

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17
Q

How does DNA fuse into a recombinant molecule?

A

Restriction Endonucleases/Enzymes are the original molecular tools that enabled Recombinant DNA technology.
Restriction endonucleases/enzymes make site-specific cuts in DNA, called restriction sites.
Today there are other alternative ways to clone DNA fragments
- via recombinases and PCR-based approaches
- restriction enzymes remain key and useful tools in molecular genetics labs

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18
Q

What are the two tyoes of restriction enzymes categorized by how they cut DNA?

A

blunt-end cutting enzymes and overhang cutting enzymes

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19
Q

What is the purpose of ‘overhangs’ in re-ligation of an inert into a vector with restriction enzymes?

A

‘Overhangs’ provide ends for re-ligation
Easy to stick molecules back together by facilitating ligation so the fragments stay associated

20
Q

What is the origin of restriction enzymes?

A

Evolved as a bacterial mechanism to target and destroy infection phage DNA.
Restriction endonucleases protect bacteria from foreign DNA and bacteria protects it’s own genome against RE by methylation.

21
Q

Define restriction enzymes.

A

an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases.

Restriction enzymes cleave DNA at specific nucleotide sequences. Restriction endonucleases cleave double-stranded DNA.

22
Q

Describe the process of ‘competent’ bacteria being transformed with ligation products/

A

Vector enters bacteria via electroporation or heat shock that opens pores in the bacteria for the vector to be pulled within. Note this does have a low probability of success.
Once successful, bacteria is grown to amplify product and then the product is isolated.

23
Q

How do I know that the recombinant vector I produce carry the right gene?

A

Two selection strategies in cloning:
(1) Selection for plasmid background
- ensure that the only surviving bacteria carries a plasmid

(2) Selection for presence of an insert
- identify among surviving clones the one with recombinant plasmids (with insert)

24
Q

Describe the selection strategy for presence of the plasmid and why it has a low success rate.

A

Most bacteria do not pick up the plasmid
Successfully transformed bacteria have the plasmid- including ampicillin resistance gene.
Plate of transformed bacteria will be the plate that survives with an antibiotic (ampicillin)

25
Q

Describe the second selection strategy: blue/white selection or LacZ and the a-complementation system.
Note: insertion has a low success rate– many plasmids do not pick up the insert

A

Separates plasmids with no insert from those with inserts that were successful in completing ligation.
Bacteria with the empty plasmid have a functional LacZ to produce the a subunit B-Galactosidase.
Bacteria with the insert have disrupted the LacZ gene and have no B-Galactosidase
X-Gal is broken down by B-Galactosidase into an insoluble blue compound

When X-Gal is added to a plate, bacteria with B-Gal turn blue, bacteria with the insert stay white

26
Q
A

Ncoll and BamHI

Because…
- EcoRI digestion splices the insert
- Sall digestion splices the selective marker gene Ampr
- Kpnl and BamHI do not have cuts in the plasmid backbone, neither do Ncol and Pstl

27
Q

Define genetic engineering.

A

the use of recombinant DNA technology to alter an organism’s genotype

28
Q

Define transgene.

A

genetically engineered DNA to be introduced to a genome

29
Q

Define transgenic organism

A

organism that contains a transgene

30
Q

Why are yeast a good model for transgenes?
(a) they are eukaryotic
(b) they grow quickly
(c) they are unicellular
(d) can be grown as haploids or diploids
(e) all of the above

A

all of the above

31
Q

How are genes inserted or replaced in yeast?

A

Transgene inserted via transformation of a plasmid, flanked by complementary sequences for the insertion targeted.
Inserted into the genome via homologous recombination.

32
Q

What is the importance of Agrobacterium in using Ti plasmids vectors?

A

Agrobacterium inserts a T-DNA on its Ti plasmid into the plant genome to infect it.
Modified Ti plasmids deliver transgenes into plant cells and pathogenic genes are removed.

33
Q

How are transgenic plants generated?

A

A. T-DNA inserts into the plant tissue and takes the transgene(s) with it
B. Infected tissue is propagated in a vegetative state
C. Infected tissue is cultured to form a whole plant that is heterozygous for the insertion
D. Homozygous plants can be generated by sexual reproduction

34
Q

How can transgenes be inserted in non-model organisms?

A

CRISPR

35
Q

How does CRISPR/Cas9 work?

A

(1) Guide RNA
- designed to match specific site of interest
- adjacent to PAM (NGG) sequence
(2) Cas9 protein cuts DNA at a specific site
- binds using guide RNA
- causes double stranded break
(3) DNA double stranded break is repaired by the cell
- NHEJ errors likely
- HR can insert sequence of interest by adding homologous DNA

36
Q

What is CRISPR used for?

A

(1) Inactivate a gene of interest by removing and NHEJ
(2) add genes of interest by swapping genes and HR
(3) Modify a gene/locus swapping a small section of a gene and HR
(4) add modified gene regulation by binding to promoter region and turning on/off specific genes

37
Q

What is the Sanger (dideoxy) sequencing method?

A

Copies DNA, in a similar way PCR does, using DNA polymerase primers, dNTPs, and ddNTPs with fluorescent tags.
Polymerase adds dideoxynucleiotides to DNA chain which terminates polymerization

38
Q

Describe the basic strategy for sequencing whole genomes.

A

Chromosomes are too large to be sequenced at once, so genomes are instead broken down, sequenced, and re-assembled using software.

(1) Break the genome into a small pool of overlapping fragments using multiple genome copies
(2) Sequence fragments to create “reads”
(3) Use computer algorithms to identify overlapping sequences and assemble reads together into larger continuous sequences
(4) the draft genome is then updated as gaps are filled with better sequencing needed to correct errors or work out complex regions

39
Q

What is Next Generation Sequencing (NGS)?

A

Cell-free, no cloning approach
Millions of DNA template fragments are isolated, prepared and simultaneously sequenced

40
Q

Why does sequencing usually require multiple copies of the same fragment?

To tell real signal from noise
To produce a signal bright enough to detect
to reduce the impact of synthesis mistakes
all of the above
none of the above

A

All of the above

41
Q

Bioinformatic-derived data helps annotate the genome by piecing out the relevant information. What is this information?

A

Protein coding genes (exons, introns, splicing sites, etc.)
Non-protein coding genes (regulatory RNAs)
Non-genic regions
- regulatory (promoters, enhancer, TF binding sites)
- Structural roles (centromere, telomere sequences)

42
Q

You discover a novel gene in unicorns that has an identical sequence to a gene in bison, but the gene is absent in their ancestors. These two identical genes are:

A

convergent

43
Q

Compare orthologs, paralogs, and homologs.

A

Homologous genes (homologs) share a common ancestor and display significant sequence conservation

Orthologs are homologous genes that are syntenic; located in the same genetic locus in closely related species

Paralogs are homologous genes that evolved as duplicates
- genes often duplicate as genomes evolve
- arose from a gene duplication in a common ancestor

44
Q

Pseudogenes are…

A

relics that reveal evolutionary ancestry; genes which used to have function in an ancestral species but have since accumulated mutations making them not functional

45
Q

What are the key differences between human and chimps?

A

Changes in regulatory sequences (cis elements) that control the expression of genes

BIOL226 (10 decks)

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