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Dentistry Biological Science > DNA Analysis 1 > Flashcards

DNA Analysis 1 Flashcards

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1
Q

What is DEFB1?

A

Defensins form a family of microbidical and cytotoxic peptides made by neutrophils. Members of the defensive family are highly similar in protein sequence. DEFB1 encodes defensive beta 1, a microbial peptide implicated in the resistance of epithelial surface to microbial colonisation.

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2
Q

How would you test the function of DEFB1 in dental caries?

A

Clone DEFB1 gene and express protein e.g. to assay its properties (toxicity, anti-microbial activity, protein interactions)

Mutagenise DEFB1 gene e.g. to assess how this affects the hose response to infection or incidence of Caries

Measure of expression of DEFB1 in patients e.g. to test whether this correlates with Caries presentation (how many express it to see if it is associated with caries)

Identify the main cells producing DEFB1 protein e.g. immune cells, salivary gland cells

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3
Q

What are four steps followed in this lecture (steps will be put a beginning of FC)?

A
  1. Clone the DEFB1 gene and express DEFB1 protein to assay its properties
  2. Mutagenise DEFB1 gene
  3. Measure the expression of DEFB1
  4. Identify the main cells producing DEFB1
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4
Q

1

Give some information on genes

A

Inheritable genetic unit, piece of DNA or RNA that encodes an RNA or a polypeptide.
DNA encodes genes.
DNA forms through pairing of purine and pyrimidine nucleotides.

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5
Q

1

Give the three types of genetic material

A

Plasmid DNA - 4 genes
E.Coli genome - 4000 genes
Human genome - 30,000 genes

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6
Q

1

Explain what gene expression is

A

RNA polymerase bind to an initiation signal at the start of the coding region and synthesise a new complementary RNA strand using free ribonucleoside triphosphate molecules (rNTP’s).
This is unwound and transcribed to make RNA. This is the functional bit of the gene needed for the gene manipulation.

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7
Q

1

How do prokaryote genes function?

A

RNA is encoded by a single open reading frame in the gene
RNA polymerases bind to the promoter on the antisense strand and synthesise a new sense strand RNA molecule
The RNA molecule is then translated.

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8
Q

1

Why is eukaryotic gene expression complex?

A

Splicing has to occur.

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9
Q

1

How do we study gene function?

A

Cloning genes and analysing their sequence and function
Analysing the expression of genes (when, where, quantity)
Test whether a gene is regulated by a specific biochemical process
Ask if a gene is involved in a gene network implicated with a disease or a biological process.

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10
Q

1

What does gene cloning involve?

A

Involves moving a gene into a vector to propagate it and over-express it.
Vectors include plasmids and viruses.
Need to move gene into plasmas so it is in the correct position and orientation for gene transcription relative to the promotor.

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11
Q

1

How does restriction endonuclease cloning work?

A

Restriction endonuclease recognise and cut site-specific stretches of dsDNA sequence (4 or more bp) to produce sticky (overhanging) or blunt edges
Restriction sites are often palindomic sequences (the same sequence on both strands)
They are made by various bacteria to digest viral DNA and many hundreds are commercially available e.g. EcoRI is derived from E.coli.

  • Sticky end produced by EcoRI digest
  • Overlapping sticky ends form hydrogen bonds between complimentary bases
  • DNA ligase binds the phosphate/sugar backbones of the two fragments together.
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12
Q

1

What do plasmid vectors have many restriction sites for?

A

Subcloning

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13
Q

1

How do we isolate the pieces of DNA for subcloning?

A

Gel electrophoresis

  1. Restriction enzymes cleave DNA into smaller segments of various sizes
  2. DNA segments loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes
  3. When an electric current is passed through the chamber, DNA fragments move towards the + charged cathode
  4. Smaller DNA segments move faster and further than larger ones
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14
Q

1

What are the 4 main cloning methods?

A

Restriction enzyme
DNA recombination
Gateway cloning
Gibson cloning

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15
Q

1

Explain the propagation of plasmids in bacteria

A

Transformation of bacteria:
DNA is added to chemically treated bacteria and mixed on ice
A shock is applied to the bacteria and they take up the DNA (e.g. 37 degrees, 45s)
The cells are allowed to recover briefly and are then plated onto a solid agar surface containing a selective antibiotic

Have to treat the bacteria so that they are able to take up the plasmid.

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16
Q

What are 4 problems with restriction enzyme cloning?

A
  1. Requires the presence of suitable restriction sites
  2. The same sites are often used in many constructs so there are less and less to choose from if you pasting together several pieces of DNA
  3. Some sites are not recognised when the plasmid is grown in certain strains of bacteria (methylation)
  4. Not all enzymes are very effective at cutting DNA
17
Q

1

Explain gene cloning by DNA recombination

A

Principle that complementary DNA sequences recognise and bind to each other.
Homologous strand recombination is mediated by recombinase enzymes that cut dsDNA and mediate a swap of homologous DNA strands e.g. recombinase enzymes from bacteriophages.

18
Q

1

What are 3 benefits of gateway cloning of genes?

A

Allows easy and rapid movement of genes (and regulatory elements) into vector of choice.
Cloning is independent of restriction endonuclease sites.
Recombination can be achieved between large (>1000kb) DNA constructs.

19
Q

1

Give three points on gibson cloning of genes

A

Allows cloning of any target sequence into vector of choice.
Cloning is mostly independent of restriction endonuclease sites (with some limitations).
Recombination does not require specialised recombination sites (or expensive enzymes).

20
Q

1

Analysing recombinant clones?

A

Can use recombinant enzymes to analyse clones to check they show the expected digest pattern.
Alternatively can sequence DNA or test it by PCR to show it is the correct piece of DNA.

21
Q

1

Explain cDNA versus genomic DNA

A

Genomic DNA encodes all genes in an organism and includes larges stretched of non-coding DNA

Gene transcription from the site in a gene generates an immature RNA that is spliced to a mature form (mRNA)

mRNA transcripts include the coding sequence for a protein and 5’ and 3’ untranslated (UTR) regulatory regions

22
Q

1

How do you copy a human gene into a bacteria to propagate it if it contains multiple exons?

A

We do it by using cDNA as this can easily be cloned into a plasmis rather than normal DNA.

23
Q

1
What is cDNA?

How do we get it to double stranded DNA?

A

Generated from an mRNA molecules by the process of reverse transcription
Often used to generate cDNA from a pool of mRNA isolated from a tissue

(isolate RNA and reverse transcribe it) End up with double stranded DNA with some sequence as original mRNA which can then go into the plasmid and be manipulated.

24
Q

1

What does the cDNA library capture? What does it involve?

A

Captures all gene expression in a tissue at a given time. Involves reverse transcribing all mRNA from a tissue/cell into cDNA and then cloning these cDNA molecules into vectors for isolation and further characterisation.

25
Q

1
How can we get enough DNA for cloning and hybridisation?

How much required DNA can be obtained per cell?

A
  • Often require micrograms quantities for cloning.
  • The coding region of a gene can be contained in a stretch of DNA that is 1-6Kbp
  • A mammalian genome is 2600Mbp
  • Typical mammalian cell has approx 3-8pg DNA per cell

you will obtain 1 x 10^-14 to 1 x 10^-13 g required DNA per cell

26
Q

1

What does PCR use and what type of reaction is it?

A

Uses a thermostable DNA polymerase to generate many copies of an original DNA template. This is an in vitro enzymatic reaction.

27
Q

1

What are the 2 principles that PCR relies on?

A
  1. Nucleic acid hybridisation: complementary nucleic DNA strands will form hydrogen bonds with each other
  2. Repetitive generation of multiple copies of a specified DNA fragment using a thermostable DNA polymerase
28
Q

1

What can we use to target a sequence of interest?

A

Sequence-specific oligonucelotides.

-Targets a defined region of DNA using specific DNA oligonucleotides primers that bind to each end (only need to know the end sequence and can amplify from a complex pool of DNA).

29
Q

1

Give details on how amplification of the target DNA is exponential

A
  • DNA oligonucleotide primers will also bind to the target sequence so they can be used for multiple amplitude events
  • Aim is to generate large copy numbers by using iterative cycles of amplification (can amplify trace amounts of DNA from samples)
  • Each cycle will double the number of copites
  • A typical reaction will have 30 cycles (2^30 fragments)
30
Q

2

Give details on the mutagenesis of a DNA sequence

A

No microbial activity

Mutation can be used to see if any affect has occurred on the cell.

Can add something to the end of the DNA or delete pieces. The primers at the end do not recognise the receptors on the DNA so that they do not incorporate.

31
Q

2

Explain substitutions, deletions and insertions when mutagenesising

A

Substitution and deletion primers are slightly modified so they have a part which is complementary to the DEFB1 gene (at the start of the gene) but a little tail which is not complementary.
Insertion primers have a base which arent complementary but that part is incorporated into the final mutated DNA.

The ones which delete have a tail that isnt complementary which blocks part of the DNA from being transcribed.
The primers which substitute only change one base in the tail of the primer.

32
Q

3

What are 3 varieties of PCR used to see if the gene has been expressed?

A
  1. Reverse transcriptase of mRNA to make cDNA followed by PCR
  2. Quantitative PCR to measure relative amounts of DNA template
  3. Quantitative PCR to measure the relative amounts of cDNA (made from mRNA)
33
Q

3

What does quantitative PCR tell us?

A

HOW MUCH DNA WE AMPLIFIED TO SEE HOW MUCH RNA WE HAD IN THE FIRST PLACE.
Measures the production of amplified DNA as the reaction occurs, not at the end as in standard PCR reactions.
Measures a target sequence by comparing to a standardised control piece of DNA.

34
Q

3
What are the two things that quantitative PCR can use?

What is measured?

A
  1. A fluorescent dye that intercalates with DNA
  2. Fluorescently labelled sequence specific oligonucleotides.

Fluorescence is measured at end of each cycle to visualise the amount of DNA product.

35
Q

3

Quicker the DNA is amplified, the —- DNA we had at the beginning

A

More

36
Q

3

Give 3 uses of PCR

A
  1. Gene cloning and mutagenesis
  2. Measuring gene expression levels
  3. Diagnostics (crime, disease, pathology, genetic screening)
37
Q

4

Explain the generation of an antisense riboprobe

A
  • Requires a template DNA with the same sequence as the target RNA
  • Use a bacteriophage RNA polymerase to synthesise an antisense RNA from a template
  • Add RNA polymerase to linearised DNA plus nucleotides
  • By adding digoxygenin-labelled rNTPs the synthesised RNA now has a label on it that can be detected
38
Q

4

What are warning with using in situ hybridisation to examine gene expression?

A
  1. Time consuming and requires a large amount of tissue
  2. Changes in the levels of expression over time cannot be quantified
  3. Relative levels of gene expression for multiple genes in a tissue cannot be compared
39
Q

Main summary points of the lecture:

  • Gene cloning involves moving a piece of DNA encoding a gene into a
  • — so that it can be propagated in bacteria
  • cDNA is generated from mRNA by reverse transcription and encodes an —- —- —- lacking any introns
  • genomic DNA is packaged into —- and encodes genes
  • PCR can hugely —- the quantities of starting DNA material
  • Q-PCR provides a quantitative measurement for gene —- levels
  • in situ hybridisation allows you to visualise where a gene —- is present in tissue
A

vector

open reading frame

chromosomes

amplify

expression

transcript

Dentistry Biological Science (88 decks)

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