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Molecular Genetics 3 > Genetic Engineering > Flashcards

Genetic Engineering Flashcards

1
Q

What is Gene cloning?

A

The process in which the gene of interest in linked to a vector that enables its amplification and propagation as a pure population of molecules in cells

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2
Q

What is molecular cloning of DNA is important to?

A
  • Purify and amplify genes of interest
  • Obtain their DNA sequences
  • Determine the gene structure and regulation
  • Perform site-directed mutagenesis to investigate function
  • Express and purify protein for biochemical/structural analysis
  • Enable genome analysis by creating overlapping clones of genomic DNA
  • Reintroduce genes into another organism i.e. transgenesis
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3
Q

What are terminal transferases?

A

Add homopolymer tails to the ends of DNA

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4
Q

What are polynucleotide kinases?

A

Add a phosphate to the 3’ end of DNA

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5
Q

What are alkaline phosphatases?

A

Remove terminal phosphates from DNA end

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6
Q

Why do bacteria use methylases?

A

Bacteria can restrict bacteriophage infection by using the appropriate endonuclease to recognise the bacteriophage’s DNA and degrade it

However, to protect its own DNA, bacteria will express cognate methylases to modify its DNA.

Presence of methylation confers cleavage so that the bacteria does not degrade its own DNA

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7
Q

Explain the binding of BamHI to its recognition sequence

A
  • BamHI restriction endonuclease binds as a homodimer and forms a 2-fold symmetric enzyme DNA complex
  • Enzyme slides along the DNA helix
  • When the recognition sequence is encountered, the conformation is altered and results in a tighter atomic interaction
  • Tighter interaction causes the cleavage of both strands
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8
Q

What are the three types of termini are produced by Type II restriction enzymes?

A

3’ recessed ends
Blunt ends
5’ Recessed ends

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9
Q

What groups do the termini have after use of the restriction enzymes

A

5’ termini of each strand in the cleavage products have Phosphoryl group

3’ termini of each strand in the cleavage product have Hydroxyl group

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10
Q

What is the function of DNA ligase?

A

Catalyse the formation of 5’ - 3’ phosphodiester bonds in double-strand DNA molecules between juxtaposed 5’ phosphate and 3’ hydroxyl

Ligase requires a co-factor that forms a covalent intermediate with the enzyme

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11
Q

How does the concentration of DNA in ligations affect the outcome of the products?

A

Higher [DNA] results in intermolecular ligation to produce linear concatenated DNA

Lower [DNA] results in intramolecular ligation to produce circular DNA (covalently closed)

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12
Q

At what concentration of DNA would the desired insert be added into a vector DNA?

A

Low [DNA] to give a covalently closed circular products

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13
Q

What is meant by the processivity of a DNA polymerase?

A

The ability of DNA polymerase to carry out continuous DNA synthesis on a template DNA without frequent dissociation.

It can be measured by the average number of nucleotides incorporated by a DNA polymerase on a single associated/dissociation event

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14
Q

Name the types of DNA polymerase (4)

A

E.coli DNA polymerase: has both 3’ to 5’ and 5’ to 3’ exonuclease activity. Has 5’ to 3’ synthesis activity

Klenow sub-fragment of DNA polymerase: lacks 5’ to 3’ exonuclease thus used for initiating DNA synthesis from oligonucleotide primers for radioactive labelling or chain terminator DNA sequence

T4 DNA polymerase: has 5’ to 3’ synthesis activity. High levels of 3’ to 5’ exonuclease activity thus useful for trimming restriction fragment ends

T7 DNA polymerase: has very high processivity and is used for chain terminator DNA sequencing

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15
Q

How is Klenow DNA pol used to make radioactive DNA probes (3)?

A
  1. The DNA molecules is denatured and hexanucleotide primers are annealed
  2. Klenow DNA pol and radioactive dNTPs are used to synthesise a labelled DNA strand until it reaches the next primer
  3. DNA is denatured and the probes are separated into their respective fragments
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16
Q

What is the function of T4 polynucleotide kinase?

A

Catalyse the transfer and exchange of phosphate from the gamma position of ATP to the 5’ hydroxyl terminus of double and single-stranded DNA and RNA

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17
Q

How can polynucleotide kinases be used?

A

To efficiently label the 5’ end of restriction fragments or synthetic single-strand oligonucleotides to use as probes using γ-32P-dNTP as the radioactive nucleotide

To phosphorylate PCR products made with non-phosphorylated primers ready for ligation

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18
Q

What are the common properties of a cloning vector?

A
  • Ability to promote autonomous replication and amplify from a single copy
  • A genetic marker(s) to select for or identify cells containing the vector
  • Unique restriction sites to facilitate cloning of insert DNA
  • Minimal non-essential DNA to maximise size range of cloning
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19
Q

How can self-ligation of the vector be prevented?

A

Alkaline phosphatase are used to remove phosphate groups from the vector ends. Therefore, the ligase is unable to form phosphodiester bond between the vector ends.

Vector and Insert can still ligate together because the insert ends have phosphate groups.

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20
Q

How can DNA molecules derived from different restriction enzymes be linked (4)?

A
  • Fill in or resect DNA ends with T4 DNA polymerase plus dNTPS and perform blunt end ligations
  • Add by blunt end ligation duplex linkers containing desired site and then cut with restriction enzymes to create cohesive end
  • Add by blunt end ligation adaptors with appropriate performed cohesive end (no restriction enzyme cutting then necessary)
  • Add desired restriction enzyme sites by PCR using oligonucleotide primers with restriction sites in 5’ end of oligonucleotide sequence and then cut with restriction enzymes to create cohesive ends
21
Q

Explain the lytic pathway of λ Bacteriophage

A
  • Lambda DNA circularises for replication
  • Catenane “rolled off” the λ DNA molecule
  • Ter enzymes generates staggered nicks at the Cos site
  • Genome packaged into phage heads

Sometimes this goes wrong and E.coli DNA is packaged instead

22
Q

Explain the in vitro process of λ DNA packaging into phage particles

A

Require two lambda strains -> 1 with mutation in pE (no head formation) and 1 with mutation in pD (no packaging). One set of bacteria infected with one lambda defect and one set with the other

Next, lyse the bacterial cells and take the lysates out. Mix lysates with recombinant lambda DNA (gene of interest).

They complete each other in vitro –> complete lambda structure

23
Q

How are λ converted into cloning vectors?

A

Non-essential region for lytic infection is deleted. This will allow insertions into λ of up to 10 Kbp

24
Q

How are λ replacement vector generated?

A

Non-essential region can be deleted entirely and substituted with a ‘stuffer’ region containing restriction enzymes sites to create a replacement vector

25
Q

What are cosmid vectors?

A

They are plasmid with a λ cos site, and are ligated with fragments of new DNA

Can take 35 Kbp in length of new DNA

26
Q

What concentration of DNA is used for the ligation of λ DNA and insert?

A

High [DNA] to produce large concatemers structure for in vitro packaging

27
Q

How are genomic DNA libraries constructed?

A

From cloning overlapping DNA fragments that have been generated partial digestion of the genomic DNA with a restriction enzyme

28
Q

What is chromosome walking?

A

Several clones can be detect with the same prove and can be aligned. Terminal regions of these clones then can be used to identify new probes to screen library to identify further clones with overlapping inserts

29
Q

What are BAC Vectors?

A

Bacterial Artificial Chromosome

They are engineered derivatives of E.coli F factors and replicate at 1-2 copies per cell with stable maintenance of their cloned inserts

They are best option for construction of genomic libraries

30
Q

Explain the production of cDNA?

A
  • mRNA is hybridised with poly-T primer
  • Make complementary DNA copy with reverse transcriptase
  • Degrade RNA with RNase H
  • Synthesise a second cDNA strand using DNA polymerase RNA fragment acts as primer
31
Q

Explain the process of adding restriction enzyme sites using linkers to improve cloning frequency

A
  1. Methylate the cDNA at GAATTC using EcoRI methylase
  2. Ligate EcoRI linkers to ends
  3. Cut with EcoRI
32
Q

How to screen cDNA library for specific clones (4) ?

A
  1. By hybridisation: DNA/RNA oligonucleotide probes
  2. By PCR
  3. Using antibodies to expressed proteins
  4. Functional cDNA screens in appropriate cell types
33
Q

What is a shuffle vector?

A

A plasmid containing two separate replication origins, selection markers and promoters for use in prokaryotic and eukaryotic cells for functional cDNA screening

34
Q

Explain the site-directed mutagenesis method

A
  1. Parental strands methylated at DpnI sites
  2. Denatyre parent strands and aneal oligonucleotide primers (incorporating the desired mutation)
  3. Add dNTPs and Pfu DNA polymerase
  4. Primer Extension
  5. Denature and re-anneal
  6. Add DpnI
    Only plasmids that are uncut by DpnI survive and transform E.coli
35
Q

Explain Golden Gate DNA Assembly

A

Bsa I cleaves away from their recognition site at different positions.

Positioning Bsa I recognition sites flanking sequences of interest that are required to be joined can allow DNAs to be cut to create compatible staggered ends for ligation.

The result is a seamless ligation product of the new join with no Bsa I sites; Bsa I sites are left on the starting DNAs

36
Q

Explain Gibson Assembly Strategy

A

1.. Requires overlap of DNA
2. Chew-back at 50 C with T5 exonuclease
3. Anneal the sticky ends Taq ligase
4. Repair at 50 C with Phusion polymerase and Taq ligase

37
Q

What are the two original DNA sequencing methods?

A
  • Sanger Method:
    Sequencing by synthesis from a primer radioactive label and base-specific termination by dideoxynucleotides
  • Chemical Degradation
    By base-specific chemical cleavage of radioactively 5’ end-labelled DNA
38
Q

What are dideoxynucleotides?

A

ddNTP have their -OH group replaced with an -H group.
Therefore, the ddNTP cannot be extended from as a phosphodiester bond cannot be formed between 3’ OH and 5’ Phosphate group

39
Q

What are the common principles of next-generation (NGS) technologies?

A
  • All rely on methods that sequence DNA DIRECTLY
  • Genomic DNA is reduced to millions of small random DNA fragments representing all the sequences of the starting DNA and these are simultaneously sequenced
  • DNA fragments are immobilised
40
Q

What is a ‘polony’ or ‘cluster’?

A

Each of the reactions occurs on a discrete solid phase clonal amplification of the single DNA molecules that were attached to the solid structure.

41
Q

What are the steps involved in the Illumina NGS method?

A
  • Sample preparations
  • Cluster generation to amplify individual DNA molecules in situ
  • Sequencing by synthesis with simultaneous imaging to record fluorescent emission
  • Data analysis
42
Q

Explain the sample preparation step of Illumina

A

Short single-strand oligonucleotides of two defined sequences (P5 and P7) are chemically bonded to the glass slides in a flow cell.

They are chemically bonded via their 5’ ends with their 3’ OH ends free pointing up

42
Q

Explain the sample preparation step of Illumina

A

Short single-strand oligonucleotides of two defined sequences (P5 and P7) are chemically bonded to the glass slides in a flow cell.

They are chemically bonded via their 5’ ends with their 3’ OH ends free pointing up

43
Q

Explain the cluster generation step of Illumina

A
  • DNA population is denatured and binds to the bonded oligonucleotides. These act as primers
  • DNA Polymerase and dNTPs are flushed throughout the flow cell and each single DNA molecule is copied starting from the 3’ end of the chemically bonded oligonucleotide primers P5 and P7
  • Denaturation and then wash away the original DNA molecule
  • Place the molecules under renaturation conditions. Anneal to adjacent chemically bonded oligonucleotide P5 primers
44
Q

Explain the sequencing and recording step of Illumina

A

Uses dNTPs with different fluorescent groups for dATP, dTTP, dCTP and dGTP, and a reversible 3’ chain blocker

Both the fluorescent group and the 3’ chain terminator block can be chemically removed after each polymerisation step

DNA polymerase is added and sequencing is started on all the single strand templates in the cluster using the reversible dNTP terminators –> dNTP terminators are incorporated

45
Q

DNase I hypersensitive sites (heterochromatin vs. euchromatin)

A

DNase hypersensitivity identifies open genomic regions in chromatin that facilitate transcription factor (TF) binding to chromatin and induce gene expression.

Nucleosomes are compacted in closed, inactive heterochromatin but are more open, exposing sites of DNase hypersensitivity in the euchromatin state.

46
Q

How does DNase-Seq work?

A

Sites of hypersensitivity (HS) are susceptible to cutting by DNase I, which releases many fragments of variable length from each hypersensitive region.

The released fragments are then purified, sequenced on one end, and the resultant sequence tags then mapped back to the genome.

A peak detection algorithm is used to identify DHS peaks

47
Q

What is Chromatin Immunoprecipitation?

A

Method of gene expression analysis:
- Fixed tissue is sonicated to shear small bits of DNA off then protein bound to DNA is immuno-precipitated with an antibody

 - Quantify DNA bound to that protein with PCR or microarray analysis

Molecular Genetics 3 (5 decks)

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