week 3- studying gene expression

Question 1

do all cells in the body share the same genome

Answer 1

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

Question 2

what are some techniques for protein analysis

Answer 2

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

Question 3

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

Answer 3

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

Question 4

why are sticky ends better

Answer 4

· 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

Question 5

how does gel electrophoresis work

Answer 5

· 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

Question 6

what are the 3 steps of recombinant DNA

Answer 6

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.

Question 7

what is the ori sequence

Answer 7

An origin of replication site which promotes replication in bacteria

Question 8

what is AMP and what is it purpose

Answer 8

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

Question 9

what is GFP

Answer 9

green flourencet protein so we can see the cultures

Question 10

steps of PCR

Answer 10

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

Question 11

what is one application of recombinant DNA and molecular biology

Answer 11

HIV

Question 12

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

Answer 12

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

Question 13

what can a genomic DNA library be used for

Answer 13

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

Question 14

what can a cDNA library contain

Answer 14

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

Question 15

what is the overall simplified version for a cDNA library

Answer 15

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

Question 16

what are the steps to make a genomic library

Answer 16

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

Question 17

what is RNA-seq

Answer 17

RNA sequencing

Question 18

what are the steps for RNA-seq

Answer 18

• 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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Question 19

how can gene mutations effect the protein function of a protein

Answer 19

.- loss of function
-gain of function

Question 20

what is a lost of function for a protein

Answer 20

-lead it to become inactive

Question 21

what is gain of a function for a protein

Answer 21

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.

Question 22

how is crispr used

Answer 22

• 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

Question 23

what is microRNA

Answer 23

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

Question 24

what can short interfering RNAs (siRNAs) do

Answer 24

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

Question 25

what is used to analyise a protein

Answer 25

SDS-PAGE (polyacrylamide gel electrophoresis)

Question 26

what are the steps of SDS-PAGE

preparation
loading
electrophoresis
reduction
visulisation
analysis

Answer 26

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.

Question 27

what is SDS-PAGE used for

Answer 27

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.

Question 28

What is PAGE in the context of protein analysis?

Answer 28

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.

Question 29

How do polypeptide chains migrate through the gel in PAGE?

Answer 29

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.

Question 30

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

Answer 30

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.

Question 31

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

Answer 31

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.

Question 32

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

Answer 32

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.

Question 33

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

Answer 33

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

Question 34

What is indirect immunohistochemistry?

Answer 34

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

Question 35

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

Answer 35

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

Question 36

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

Answer 36

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

Question 37

How is a GFP-fusion protein created?

Answer 37

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.

Question 38

What is RNA interference (RNAi)?

Answer 38

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

Question 39

What are microRNAs and what is their role in RNAi?

Answer 39

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

Question 40

How can we use RNAi technology?

Answer 40

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

Question 41

How can RNAi be engineered for specific uses?

Answer 41

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

Question 42

What is a homozygous mutant?

Answer 42

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

Question 43

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

Answer 43

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.

Question 44

What is RNA-Seq?

Answer 44

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

Question 45

How does RNA-Seq help in gene expression studies?

Answer 45

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.