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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

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
Q

What is a genomic library?

A

A collection of clones that contains at least one copy of every DNA sequence in a genome

26
Q

How can a fragment of DNA be studies using a genomic library?

A

A clone containing that DNA can be isolated from the library by using a specific probe.

27
Q

What is a cDNA library?

A

A collection of clones representing at least one copy of each of the genes expressed in a cell

28
Q

How are cDNA libraries made?

A

mRNA is isolated from eukaryotic cells and cDNA is synthesized by reverse transcriptase using the mRNA as a template

29
Q

How is a cDNA library different from a genomic library?

A

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
Q

What is an expression library?

A

cDNAs inserted into an expression vector. These can be inserted into a cell for transcription and translation into protein inside the host cell.

31
Q

What is used as a priming site for cDNA synthesis?

A

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
Q

What are the steps of cDNA synthesis?

A
  • 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
Q

What three enzymes are needed for cDNA synthesis?

A
  • Reverse transcriptase
  • Ribonuclease H
  • DNA polymerase I
34
Q

How are libraries screened for hybridization? (2 methods)

A

With DNA or RNA probes

  • Colony hybridization
  • Plaque hybridization

These methods similar to southern/northern blotting

35
Q

What type of screening can be used for expression libraries? What is the advantage of this method?

A

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
Q

How many distinct primers are needed for PCR?

A

A pair of oligonucleotides (2)

37
Q

What are the three steps of PCR?

A
  • Denaturation
  • Annealing
  • Extension
38
Q

What are two molecular biology applications for PCR?

A
  • 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
Q

What is asymmetric PCR and how is it done?

A
  • Synthesis of single stranded DNA

- Only one of the two PCR primers is used

40
Q

What is RT-PCR? How is it done?

A

Reverse transcriptase PCR

  • Synthesized cDNA from RNA using reverse transcriptase
  • cDNA is used as template for PCR
41
Q

What are three common problems with PCR?

A
  • 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
Q

What is a contig? What technique is used to make it?

A

A contiguous set of overlapping DNA sequences. Made with chromosome walking

43
Q

Why do we want to make a contig and use chromosome walking?

A

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
Q

What is an electrophoretic technique that allows for the separation of very large DNA molecules and intact chromosomes?

A

Pulsed-field gel electrophoresis (PFGE)

45
Q

Why is pulsed field gel electrophoresis necessary for large molecules of DNA?

A

Because molecules of DNA above 30-50 kb migrate with the same mobility regardless of size

46
Q

What is an essential step for chromosomal DNA that is not needed for non-chromosomal DNA?

A

Chromosomal DNA must be treated with proteases to remove proteins. Especially things like histones.

47
Q

What is a restriction fragment length polymorphism (RFLP)?

A

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
Q

What are simple sequence length polymorphisms?

A

Arrays of repeat sequences that display length variations, with different alleles containing different numbers of repeat units.

49
Q

What is a difference between restriction fragment length polymorphisms (RFLPs) and simple sequence length polymorphisms (SSLPs)?

A

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
Q

What are two types of simple sequence length polymorphisms?

A
  • Minisatellites - variable number of tandem repeats (VNTR)

- Microsatellites (simple tandem repeats - STRs)

51
Q

What are minisatellites (variable number of tandem repeats) SSLPs?

A
  • Containing repeat units tens of nucleotides in length

- Non-randomly distributed around the genome (tend to be near chromosome ends, eg. telomeric repeats)

52
Q

Where does variation in minisatellites come from?

A

Unequal crossing over during meiosis

53
Q

What are microsatellites (simple tandem repeats - STRs)?

A
  • Di, tri or tetra nucleotide repeat units
  • Randomly distributed in genomes
  • Shorter than VNTRs
54
Q

How are microsatellites (STRs) generated?

A

Slippage during DNA replication

55
Q

How can simple sequence length polymorphisms be detected by PCR?

A

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
Q

What are single nucleotide polymorphisms (SNPs)?

A

Point mutations carried by some individuals of a population

57
Q

How are cloned DNA molecules induced to enter host cells?

A

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
Q

How is the origin or replication (ori) on BACs and YACs different from that of plasmids?

A

It regulates for slower copying so that the cell isn’t burdened by the size of the plasmid

59
Q

Why do YACs need telomeric and centromeres?

A

Centromeres are for mitosis separation and telomeres are needed for replication as well.

60
Q

How can RFLPs be used to identify a DNA sample?

A

Can analyze them with southern hybridization to determine if DNA sample is from someone.

61
Q

What type of polymorphism is assayed in paternity/fraternity etc. tests?

A

SSLP (simple sequence length polymorphisms)

62
Q

What type of polymorphism accounts for the most amount of variation among people?

A

Single nucleotide polymorphisms (SNPs), can be predicted by observing microsatellites (simple tandem repeats)

Nucleic Acid Biochem II (8 decks)

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