week 3- studying gene expression Flashcards

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1
Q

do all cells in the body share the same genome

A

yes, just expression of genes is different in other/ different cells

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2
Q

what are some techniques for protein analysis

A

· SDS-polyacrylamide gel electrophoresis and western blotting
· Immunofluorescence / immunohistochemistry
Fusion proteins

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3
Q

what is a restriction enzyme that gives rise to a) blunt end 2) sticky end

A

Some restriction enzymes cut blunt (haelll)or stick ends (ecoRI HindIII)

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4
Q

why are sticky ends better

A

· The sticky ends are better because they leave ‘overhang’ which help to join the DNA back together through complimentary base pairs. The re-joining of DNA is important in DNA cloning

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5
Q

how does gel electrophoresis work

A

· Separated DNA from size
· Different restriction enzymes cut at different sites, thus the fragments are separated
· The negative DNA will migrate to the postive electrode
The lower bands are closer to the positive electrode

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6
Q

what are the 3 steps of recombinant DNA

A

1) transform e. coli with the recombinant plasmid where some are taken up, none are taken up
2) plate bacteria with antibiotic resistance
3) Isolate multiple individual colonies and amplify each colony in a separate flask.

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7
Q

what is the ori sequence

A

An origin of replication site which promotes replication in bacteria

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8
Q

what is AMP and what is it purpose

A

For bacterial resistance to ampicillin, this ensure that only bacteria which contain the plasmid will grow on culture plates containing ampicillin

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9
Q

what is GFP

A

green flourencet protein so we can see the cultures

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10
Q

steps of PCR

A

1) heat DNA to 96 degrees to seperate the 2 DNA strands by denatureing the hydrogen bonds
2) sample is cooled to anneal the primers
3) Taq polymerase is added to synthesise a complementary DNa sequence
4) the process is completed until stopped it runs out of bases

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11
Q

what is one application of recombinant DNA and molecular biology

A

HIV

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12
Q

what are the tools to use molecular biology to understand the HIV virus

A
  1. blood sample with a person infected with HIV
  2. extract the RNA from the HIV particle in the plasma of the infected person
  3. use reverse transcription and PCR of the cDNA
  4. put this into gel electrophoresis agaisnt a person who is not infected to see the BP of the virus
  5. put the sample in a plasma vector (recombinate DNA) to overexpress the protein
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13
Q

what can a genomic DNA library be used for

A

A genomic DNA library can be used to determine the nucleotide sequence of an entire genome.

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14
Q

what can a cDNA library contain

A

A cDNA library contains clones at a frequency that mirrors the frequency of mRNA molecules in tissues.

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15
Q

what is the overall simplified version for a cDNA library

A

We can isolate DNA and fragment into plamids to create a library

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16
Q

what are the steps to make a genomic library

A
  1. double stranded DNA is cleaved with restriction enzymes, crating millions of DNA fragments
  2. DNa fragments are inserted into plasmids
  3. these fragments are introduced into bacteria which creates a genomic bacteria
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17
Q

what is RNA-seq

A

RNA sequencing

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18
Q

what are the steps for RNA-seq

A
  • Extract and fragment RNA from cells/tissue/sample of interest
    • Synthesise cDNA (convert mRNA to cDNA)
    • Adapter ligation (add adapters to ends of cDNA fragments)
    • PCR amplification of sequences of interest
    • Select fragments of desired size
    • Sequence
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19
Q

how can gene mutations effect the protein function of a protein

A

.- loss of function
-gain of function

20
Q

what is a lost of function for a protein

A

-lead it to become inactive

21
Q

what is gain of a function for a protein

A

can lead to protein activity in the absence of a normal activation stimulus and can cause, for example, tumor growth, in the case of abnormal activation of an oncogene.

22
Q

how is crispr used

A
  • Preforms precise gene editing
    • Cas9 protein binds to a guide RNA. The proportion if RNA is used for associated to the cas9 protein
    • The guide RNA helps the complete find sequence sequences so it can be precise
      The PAM sequence- is cleaved. Helps the complete to distinguish its own DNA and viral DNA
23
Q

what is microRNA

A

. microRNAs are non-coding RNAs that degrade or block translation of specific mRNAs.

24
Q

what can short interfering RNAs (siRNAs) do

A

short interfering RNAs (siRNAs) to repress or inhibit gene expression.

25
Q

what is used to analyise a protein

A

SDS-PAGE (polyacrylamide gel electrophoresis)

26
Q

what are the steps of SDS-PAGE

preparation
loading
electrophoresis
reduction
visulisation
analysis

A

Protein Preparation: Proteins are treated with SDS, making them negatively charged and unfolded.

Loading onto Gel: The unfolded proteins are placed into wells in a gel made of polyacrylamide, which acts like a sieve.

Electrophoresis: An electric current is applied, causing the proteins to move through the gel based on their size; smaller proteins move faster.

Reduction: A reducing agent, like β-mercaptoethanol, breaks disulfide bonds between protein subunits, allowing each subunit to be analyzed separately.

Visualization: Proteins are stained to be visible as bands on the gel.

Analysis: Scientists examine the size and amount of each protein band to understand the protein mixture.

27
Q

what is SDS-PAGE used for

A

technique can be used to determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a protein. For example, a protein with two subunits will run as two separate bands, based on their molecular weight, in the same lane.

28
Q

What is PAGE in the context of protein analysis?

A

PAGE stands for polyacrylamide gel electrophoresis, a technique where prepared protein samples are loaded onto a polyacrylamide gel housed in an electrophoresis apparatus containing buffers.

29
Q

How do polypeptide chains migrate through the gel in PAGE?

A

Polypeptide chains form complexes with negatively charged SDS molecules and migrate as negatively charged SDS-protein complexes through the porous gel. SDS and β-mercaptoethanol treatment breaks disulfide bonds, allowing each subunit to be analyzed independently.

30
Q

How does PAGE help determine the molecular weight of polypeptide chains?

A

The speed of migration is inversely proportional to the size of the polypeptide, allowing determination of approximate molecular weight and subunit composition. A protein with two subunits will appear as two separate bands based on molecular weight.

31
Q

What can be done after proteins are separated on the gel?

A

The separated proteins can be transferred or ‘blotted’ onto a membrane, which can be stained to visualize total protein or incubated with antibodies to detect specific proteins.

32
Q

How can the purity of an enzyme be assessed using PAGE and staining?

A

By analyzing lanes on a Coomassie-stained gel at successive stages of purification, you can see the relative purity of the enzyme. For example, lane 5 may show a pure fraction of the enzyme with a molecular mass of around 40kDa.

33
Q

What marker molecules are used in indirect immunohistochemistry and what are they used for?

A

Fluorescent probes (for fluorescence microscopy)
Horseradish peroxidase enzyme (for light or electron microscopy)

34
Q

What is indirect immunohistochemistry?

A

Indirect immunohistochemistry is a sensitive detection method where many secondary antibody molecules recognize each primary antibody . The secondary antibody is coupled to a marker molecule that is detectable.
LOOKS FOR THE CONSTANT REGION ON THE ANTIBIDY WHICH IS THE SAME FOR ALL ANTIBODIES

35
Q

How can different fluorescent probes be visualized in the same cell?

A

Different fluorescent probes can be visualized in the same cell by labeling different cellular components with specific probes. For example, in a cell in mitosis:

Green fluorescent antibody labels spindle microtubules
Red fluorescent antibody labels centromeres
Blue fluorescent dye (DAPI) labels the DNA of condensed chromosomes

36
Q

What are GFP tagged proteins or ‘fusion proteins’ used for?

A

GFP tagged proteins or ‘fusion proteins’ are used to visualize proteins in living cells.

37
Q

How is a GFP-fusion protein created?

A

The GFP coding sequence is inserted at the beginning or end of the gene of interest, creating a GFP-fusion protein that can be easily visualized.

38
Q

What is RNA interference (RNAi)?

A

RNA interference (RNAi) is a simple and fast method to investigate gene function by inhibiting gene expression.

39
Q

What are microRNAs and what is their role in RNAi?

A

MicroRNAs are non-coding RNAs that degrade or block the translation of specific mRNAs, thereby regulating gene expression.

40
Q

How can we use RNAi technology?

A

We can design and introduce short interfering RNAs (siRNAs) to repress or inhibit the expression of specific genes.

41
Q

How can RNAi be engineered for specific uses?

A

RNAi technology can be engineered to create cell-, tissue-, and time-specific RNA interference, allowing precise control over gene expression.

42
Q

What is a homozygous mutant?

A

A homozygous mutant is an organism that has two identical mutant alleles for a specific gene.

43
Q

What does it mean for an organism to be homozygous for a mutant allele?

A

being homozygous for a mutant allele means the organism inherited the same mutant version of a gene from both parents, which can lead to changes in its observable characteristics or phenotype.

44
Q

What is RNA-Seq?

A

RNA-Seq is a technique used to analyze the transcriptome, providing information about the quantity and sequences of RNA transcripts in a sample

45
Q

How does RNA-Seq help in gene expression studies?

A

RNA-Seq quantifies the abundance of RNA transcripts, helping researchers understand which genes are expressed and at what levels under various conditions, providing insights into gene function and regulation.

46
Q
A