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ESA 1 Medical Cell Biology And Genetics (BG) > Molecular Techniques > Flashcards

Molecular Techniques Flashcards

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1
Q

Requirements for gel electrophoresis: (4)

A

Gel - matrix allowing separation of DNA

Buffer - Allows charge on the DNA samples across the gel

Power supply - Generates charge difference across the gel

Stain/detection - to identify the presence of the separated DNA

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2
Q

What are restriction enzymes?

A

Molecular scissors.

They recognise and cut specific DNA sequences. Mainly palindromes. Come from bacteria

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3
Q

Why do we use restriction analysis?

A

To investigate the size of DNA fragments

To investigate mutations

To investigate DNA variation

To clone DNA

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4
Q

What are plasmids?

A

Small circular dsDNA

Found in bacteria

Mini chromosomes

Carry genes to replicate independently

Can transfer to other bacteria

Often carry antibiotic resistance genes

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5
Q

Four basic steps of gene cloning:

A

Isolate relevant gene of interest following digestion with restriction enzymes

Insert gene of interest into plasmid vector

Introduce recombinant DNA molecule into suitable host cells

Identify and isolate clone containing DNA of interest

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6
Q

Why do we clone human genes?

A

To make useful proteins (insulin)

To find out what genes do

Genetic screening

Gene therapy

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7
Q

Steps of PCR:

A

Denaturation - DNA heated to 95 degrees

Annealing - the two primers bind the appropriate complimentary strand

Primer extension - DNA polymerase extends the primer. Taq polymerase works best at 72 degrees.

(Repeated 28-35 times)

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8
Q

Why use PCR?

A

To amplify a specific DNA fragment

To investigate single case mutations

To investigate small deletions or insertions

To investigate variation, genetic relationships

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9
Q

How are the proteins separated in electrophoresis?

A

Proteins are charged molecules and move towards the anode or cathode if placed in an electric field

Proteins are separated on basis of size, shape or charge

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10
Q

How is DNA separated in electrophoresis?

A

DNA is negatively charged and will move towards the anode of placed in an electric field

DNA fragments can be separated on the basis of size (or shape)

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11
Q

What does SDS-PAGE do?

A

Separation of proteins on the basis of size

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12
Q

What is Isoelectric focusing (IEF)?

A

Proteins separate on basis of charge

Proteins migrate until they reach a pH equal to their pl

No net charge at pl so stop migrating

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13
Q

Steps of Isoelectric focusing (IEF)

A

A stable pH gradient is established in the gel after application of an electric field

Protein solution is added and electric field is reapplied

After staining, proteins are shown to be distributed along pH gradient according to pl values

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14
Q

Overview of 2D-PAGE

A

Allows separation of complex mixtures of proteins

Important for diagnosing disease states in different tissues

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15
Q

Proteomics steps

A

Digest protein with trypsin

Perform mass spectrometry

Generate list of peptide sizes

Use database of predicted peptide sizes for known proteins to identify the protein

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16
Q

Proteomics is

A

The analysis of all proteins expressed from genome

17
Q

Molecular diagnosis is

A

Analysis of a single purified protein

18
Q

Antibodies characteristics:

A

Bind to specific protein targets (antigens)

Recognise a few amino acids on a protein (epitope)

19
Q

Monoclonal antibodies

A

Produced from 1 B Lymphocyte

I identical antibody

Specific to 1 antigen

1 epitope

20
Q

Polyclonal antibodies

A

Produces by many B lymphocytes

Multiple different antibodies

Specific to 1 antigen

Multiple epitopes

21
Q

What can an enzyme-linked immunoabsorbent assay (ELISA) be used for?

A

Can be used to measure the concentration of proteins in solution

22
Q

What is the gold standard for diagnosis of an MI?

A

Cardiac troponin I (cTnI) by ELISA

23
Q

What is southern blotting?

A

Combined gel electrophoresis and DNA hybridisation

24
Q

What is northern blotting?

A

Uses DNA to detect RNA species. Combined gel electrophoresis and DNA hybridisation

25
Q

What is western blotting?

A

Involves the detection of proteins by antibodies after protein gel electrophoresis

26
Q

Steps of southern blotting:

A

Digest DNA with restriction enzymes

Separate DNA fragments by gel electrophoresis

Transfer DNA to nylon

Hybridise filter with labelled gene probe

Detect hybridisation by exposure to x ray film

27
Q

Why do we use southern blotting?

A

To investigate gene structure

To investigate gene expansions, triplet repeats

To investigate mutations in genetic tests

To investigate variation, genetic relationships

28
Q

Characteristics of DNA proves in binding:

A

Do not need 100% similarity to target sequence

Do not need to completely align with target

Do not affect the position of the target sequence on the gel

29
Q

How does DNA sequencing work?

A

A tube contains all of the dNTPs, DNA polymerase and one of the ddNTPs.

It also has denatured DNA and a primer.

When the ddNTP is added the chain stops elongating.

This is done for all ddNTPs and then they are run on a gel and identified by labelling to read the sequence

30
Q

Why would we use RT-PCR?

A

It reveals information about gene expression

31
Q

Outline RT-PCR:

A

Nuclease breaks down the RNA to remove

DNA primers are used and anneal

Reverse transcriptase catalyses the reaction to extend the sequence

PCR is used to amplify the cDNA (complimentary)

32
Q

What is microarray technology?

A

Analysis of 1000’s genes on a slide

Can compare two conditions

Can die the two slides with different probes. You can compare gene expression depending on the size of the signal

You can see duplications and deletions

33
Q

What is chromosome painting?

A

Making proves specific for each chromosome.

Can be helpful to identify translocation and can be used for cancer cell analysis

34
Q

What is FISH?

A

Probe DNA is labelled with fluorescent dye

Target chromosome denatured and hybridised

Re annealed and can be viewed

ESA 1 Medical Cell Biology And Genetics (BG) (1 decks)

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