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Nucleic Acid Biochem II > 15-17 Recombinant DNA > Flashcards

15-17 Recombinant DNA Flashcards

216-224 202-203 224-228 252 (62 cards)

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1
Q

What is the ‘full’ definition of cloning?

A

The isolation of a particular nucleotide DNA sequence from its genomic context and the production of multiple identical copies of that sequence

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2
Q

What are three uses of cloning?

A
  • Isolation of genes or gene fragments for in vitro study
  • To obtain large quantities of a protein encoded by a particular gene for in vitro study or medical purposes (eg. insulin)
  • Perpare modified versions of genes for reintroduction into the original host for functional studies
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3
Q

What requires vectors, hosts and DNA manipulation?

A

Cloning

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4
Q

What are the broad steps of cloning? (4)

A
  • Foreign DNA fragment is prepared by restriction or PCR for insertion into a vector
  • The fragment is inserted and ligated into the vector to make a recombinant DNA molecule
  • These recombinant DNA molecule is inserted into host cells and the cell culture is heat shocked or electroporated
  • Some sort of selection for cells containing recombinant DNA molecules occurs
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5
Q

What are used for cloning relatively small inserts? (less than 10 kbp)

A

Plasmid Vectors

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6
Q

How big are plasmid vectors?

A

Around 1-200 kb in size

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7
Q

What are three essential features of plasmid vectors?

A
  • Have an origin of replication (ori) site
  • Have selectable markers (to distinguish cells with the plasmid from those without)
  • Unique restriction enzyme cleavage sites (most located in MCS)
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8
Q

What type of vectors are used to clone inserts around 20 kbp?

A

Bacteriophage cloning vectors (eg. lambda genome)

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9
Q

What type of vectors are used for 100-300+ kbp fragment cloning?

A

BACs (Bacterial artificial chromosomes)

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10
Q

BACs are propagated at high or low copy numbers in E. coli?

A

low copy numbers

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11
Q

What type of vector is used to clone ‘linear DNA fragments’ up to 2000 kbp?

A

YACs (yeast artificial chromosomes)

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12
Q

What are three components of YACs (yeast artificial chromosomes)?

A
  • Yeast replication origin (ori)
  • Centromere (CEN)
  • Telomere (TEL)
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13
Q

How do YACs come? How must they be treated to allow for fragment insertion?

A

YACs come as circular chromosomes. They contain two telomere sequences bounded internally by BamHI cleavage sites. Cleavage by BamHI makes a linear molecule with telomeres at two ends.

Then EcoRI digestion must occur to cleave X and Y markers from each other and genomic DNA is inserted and ligated between these. The recombinant YAC is then ready to transform yeast cells.

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14
Q

How are YACs inserted into yeast cells?

A
  • The yeast cell wall must be enzymatically degraded

- The resulting yeast spheroplast is then susceptible to transformation from YAC

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15
Q

What type of restriction enzymes can be used to generate cloning compatible ends?

A

Type II restriction endonucleases

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16
Q

How can PCR reactions add restriction sites to molecules?

A

By adding restriction sites to 5’ ends of the PCR primers

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17
Q

How are restriction enzyme linkers (aka adapters) added to blunt end restriction endonuclease products?

A

With T4 DNA ligase

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18
Q

What is TA cloning?

A

Takes advantage of the fact that DNA polymerases (eg. Taq polymerase) can add a single 3’ A residue to blunt ended double stranded DNA
- PCR products or other A tailed products can therefore be ligated (inserted) with DNA ligase into a T-tailed vector

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19
Q

What is a T-tailed vector?

A

A linearized vector with 3’-T overhangs

Can have A tailed products ligated (inserted) in

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20
Q

What is TOPO-TA cloning?

A

TA cloning (inserting A-tailed products into T-tailed vectors), but with a topoisomerase covalently attached to the vector. The 5’ OH group of a A-tailed PCR product can attach this bond and release the topoisomerase I enzyme.

Take home message is that DNA ligase is not needed. The vector come pre-prepared, just add the A-tailed product (from Taq polymerized PCR etc.)

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21
Q

What is one method of directional cloning?

A
  • Use a restriction enzyme that makes sticky ends for one end of the insertion and a another restriction enzyme to make a blunt end at the other end to digest chromosomal DNA for insertion
  • Can insert into a plasmid cloning vector cleaved with the same restriction enzymes
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22
Q

What type of plasmid is used in blue-white screening? What are it’s three main features?

A

A pUC8 plasmid

  • Ampicilin resistance gene
  • lacZ gene codes for beta-galactosidase enzyme
  • Has a MCS within lacZ open reading frame
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23
Q

How does the blue-white screening test work?

A

Because the pUC8 plasmid used has a MCS within its lacZ gene, those with an insert will not produce a viable beta-galactosidase. When plated with X-gal sugar, the non-recombinant host will split x-gal into components, one of which is blue. The recombinant host cannot split x-gal and therefor is white.

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24
Q

Does a blue product from blue-white screening indicate recombinance or no recombinance? Why?

A

No recombinance. Because blue product is the result of x-gal digestion by beta-galactosidase, which is not produced when a recombinant insert is in the lacZ gene of the pUC8 plasmid.

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25
What is a genomic library?
A collection of clones that contains at least one copy of every DNA sequence in a genome
26
How can a fragment of DNA be studies using a genomic library?
A clone containing that DNA can be isolated from the library by using a specific probe.
27
What is a cDNA library?
A collection of clones representing at least one copy of each of the genes expressed in a cell
28
How are cDNA libraries made?
mRNA is isolated from eukaryotic cells and cDNA is synthesized by reverse transcriptase using the mRNA as a template
29
How is a cDNA library different from a genomic library?
A cDNA library reflects gene activity of the cells at the time the mRNA was isolated, rather than all copies of DNA sequence in a genome
30
What is an expression library?
cDNAs inserted into an expression vector. These can be inserted into a cell for transcription and translation into protein inside the host cell.
31
What is used as a priming site for cDNA synthesis?
The poly(A) tails at the 3' end of mRNA molecules. Reverse transcriptase uses this sequence as a priming site to make an entirely T oligonucleotide primer.
32
What are the steps of cDNA synthesis?
- mRNA is isolated - Reverse transcriptase adds a DNA oligo(dT) primer to the 3' A tail of mRNA - REverse transcriptase synthesizes the rest of the strand of DNA - Ribonuclease H degrades most of the mRNA template - Second strand synthesis is by DNA pol I
33
What three enzymes are needed for cDNA synthesis?
- Reverse transcriptase - Ribonuclease H - DNA polymerase I
34
How are libraries screened for hybridization? (2 methods)
With DNA or RNA probes - Colony hybridization - Plaque hybridization These methods similar to southern/northern blotting
35
What type of screening can be used for expression libraries? What is the advantage of this method?
Antibody screening, where an antibody reacts with an expressed protein to identify clone of interest. Allows you to isolate cDNAs when you do not have a suitable DNA/RNA probe.
36
How many distinct primers are needed for PCR?
A pair of oligonucleotides (2)
37
What are the three steps of PCR?
- Denaturation - Annealing - Extension
38
What are two molecular biology applications for PCR?
- PRobe synthesis (perform PCR in presence of labeled nucleotides) - Cloning to add restriction sites to the 5' end of PCR primers or for Taq polymerases A-tails
39
What is asymmetric PCR and how is it done?
- Synthesis of single stranded DNA | - Only one of the two PCR primers is used
40
What is RT-PCR? How is it done?
Reverse transcriptase PCR - Synthesized cDNA from RNA using reverse transcriptase - cDNA is used as template for PCR
41
What are three common problems with PCR?
- DNA synthesis by Taq polymerase is sloppy due to lack of proofreading activity - The method is very sensitive and contamination must be avoided - Must know about the target region of DNA beforehand so that appropriate primers can be designed
42
What is a contig? What technique is used to make it?
A contiguous set of overlapping DNA sequences. Made with chromosome walking
43
Why do we want to make a contig and use chromosome walking?
Often we want to place the genomic information contained in a single cloned fragment into a larger context. Starting with the DNA sequence of a single clone, probes can be used to 'screen' a library for additional clones that contain fragments overlapping with the original clone. - So DNA that is too large to sequence in one piece is fragmented and inserted (cloned) into vectors, these inserted fragments are then sequenced one by one
44
What is an electrophoretic technique that allows for the separation of very large DNA molecules and intact chromosomes?
Pulsed-field gel electrophoresis (PFGE)
45
Why is pulsed field gel electrophoresis necessary for large molecules of DNA?
Because molecules of DNA above 30-50 kb migrate with the same mobility regardless of size
46
What is an essential step for chromosomal DNA that is not needed for non-chromosomal DNA?
Chromosomal DNA must be treated with proteases to remove proteins. Especially things like histones.
47
What is a restriction fragment length polymorphism (RFLP)?
A restriction fragment whose length is variable due to the presence of a polymorphic restriction site at one or both ends. Fragment numbers and sizes can vary between alleles of differing parental strands
48
What are simple sequence length polymorphisms?
Arrays of repeat sequences that display length variations, with different alleles containing different numbers of repeat units.
49
What is a difference between restriction fragment length polymorphisms (RFLPs) and simple sequence length polymorphisms (SSLPs)?
SSLPs can be multiallelic, meaning each SSLP can have a number of different length variants. Also, SSLPs are variable in repeat units while RFLPs are variable in restriction sites
50
What are two types of simple sequence length polymorphisms?
- Minisatellites - variable number of tandem repeats (VNTR) | - Microsatellites (simple tandem repeats - STRs)
51
What are minisatellites (variable number of tandem repeats) SSLPs?
- Containing repeat units tens of nucleotides in length | - Non-randomly distributed around the genome (tend to be near chromosome ends, eg. telomeric repeats)
52
Where does variation in minisatellites come from?
Unequal crossing over during meiosis
53
What are microsatellites (simple tandem repeats - STRs)?
- Di, tri or tetra nucleotide repeat units - Randomly distributed in genomes - Shorter than VNTRs
54
How are microsatellites (STRs) generated?
Slippage during DNA replication
55
How can simple sequence length polymorphisms be detected by PCR?
Electrophoresis bands can be compared to standard. If there are two bands, than both alleles are different SSLPs. Repeats make fragments larger (think 2030 lab)
56
What are single nucleotide polymorphisms (SNPs)?
Point mutations carried by some individuals of a population
57
How are cloned DNA molecules induced to enter host cells?
Heat shocking or electroporation bacteria disrupts cell membrane to allow fragment DNA to enter. Yeast cell membranes can be enzymatically degraded to allow transformation.
58
How is the origin or replication (ori) on BACs and YACs different from that of plasmids?
It regulates for slower copying so that the cell isn't burdened by the size of the plasmid
59
Why do YACs need telomeric and centromeres?
Centromeres are for mitosis separation and telomeres are needed for replication as well.
60
How can RFLPs be used to identify a DNA sample?
Can analyze them with southern hybridization to determine if DNA sample is from someone.
61
What type of polymorphism is assayed in paternity/fraternity etc. tests?
SSLP (simple sequence length polymorphisms)
62
What type of polymorphism accounts for the most amount of variation among people?
Single nucleotide polymorphisms (SNPs), can be predicted by observing microsatellites (simple tandem repeats)

Nucleic Acid Biochem II (8 decks)

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