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Molecular Techniques Flashcards

1
Q

What are restriction enzymes?

A

Endonucleases produced by bacteria

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2
Q

What are the functions of restriction enzymes?

A

Recognition and degradation of foreign DNA

Recognise and cut specific DNA sequences (restriction sites)

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3
Q

How do restriction enzymes cut DNA?

A

Break phosphodiester bonds between adjacent nucleotides - giving sticky ends

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4
Q

What is common in the sequences that restriction enzymes usually cut?

A

They usually cut palindromic sequences

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5
Q

What technique is used to separate DNA fragments created by restriction enzymes?

A

DNA gel electrophoresis

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6
Q

On what basis does DNA gel electrophoresis separate DNA fragments?

A

On the basis of size

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7
Q

What is required for DNA gel electrophoresis?

A

Agarose Gel - matrix that allows the separation of fragments
Buffer - allows charge on the DNA samples
Power Supply - generates charge difference across gel
Stain/Detection - to identify presence of separated DNA (UV)

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8
Q

Where do the DNA fragments in gel electrophoresis start? Why?

A

At the cathode (-ve electrode)

DNA is negatively charged due to phosphate groups moves towards the positive electrode (anode)

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9
Q

How are DNA fragments separated in DNA gel electrophoresis?

A

Larger fragments move less far in the gel

Smaller fragments move further in the gel

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10
Q

What can restriction analysis be used to investigate?

A

To investigate small deletions (size of DNA fragment)
To investigate mutations
To investigate DNA variation
To clone DNA

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11
Q

What vector is used in gene cloning?

A

Plasmid

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12
Q

What are some features of plasmids?

A

Small circular DNA found in bacteria

Can transfer to other bacteria (e.g. Antibiotic resistance genes)

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13
Q

What are the steps involved in gene cloning?

A

Isolate the DNA you want using restriction enzymes
Place DNA into plasmid using DNA ligase (producing recombinant DNA)
Introduce into suitable host cell (e.g. E. coli)
Identify and isolate the clone that contains the certain gene

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14
Q

What is a common application for gene cloning?

A

Making proteins (e.g. Insulin)

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15
Q

Are copies of genes initially taken in the RNA or DNA form? Why?

A

RNA form - doesn’t contain introns —> direct code for protein
Reverse transcriptase —> RNA —> DNA —> plasmid

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16
Q

What is PCR useful for?

A

Amplification of a target DNA sequence

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17
Q

What three things does PCR require?

A

A thermostable DNA polymerase (Taq polymerase)

Specific forward and reverse primers

Temperature cycling - denature, anneal, polymerise

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18
Q

What is PCR useful for investigating?

A

Single base mutations
Small deletions
Genetic variation

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19
Q

What are the 3 steps involved in PCR and their temperatures?

A

Denaturing (95 degrees Celsius)
Annealing (55 degrees Celsius)
Polymerisation (72 degrees Celsius)

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20
Q

What is the function of protein gel electrophoresis?

A

To separate proteins based on size, charge and shape (usually size)

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21
Q

What 4 things are required for protein gel electrophoresis?

A

Gel
Buffer
Power supply
Stain

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22
Q

How does protein gel electrophoresis work?

A

Contents of cell (mixture of macromolecules) placed at wells near cathode in a vertical gel

Larger proteins move less far in the gel
Smaller proteins move further in the gel

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23
Q

When is SDS-PAGE used?

A

When you want to ignore the intrinsic properties of a protein and separate by size only

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24
Q

How does SDS-PAGE work?

A

Protein unravelled —> using detergent; SDS and beta-ME

Then separated on a gel using protein gel electrophoresis

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25
What is isoelectric focusing used for?
To separate proteins based on their isoelectric point (charge)
26
What is the isoelectric point of a protein?
The pH at which the protein carries no charge
27
How is isoelectric focusing carried out?
Ph gradient created in a gel with an electric field | Proteins migrate till they reach the pH that equals their pI
28
What is 2D-PAGE used for?
Used to separate complex mixtures of proteins based on charge and size Important in diagnosing disease states
29
How is 2D-PAGE carried out?
Usually isoelectric focusing followed by SDS-PAGE
30
How can proteases be useful in identifying mutations?
Specific proteases cut proteins at certain amino acid residues ---> so will change the size of fragments produced
31
What is an epitope?
Part of the antigen that the antibody binds to
32
How do antibodies bind to an antigen?
Antibodies recognise a specific amino acid sequence on an antigen called the epitope
33
What are two types of antibodies?
Polyclonal antibodies | Monoclonal antibodies
34
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal antibodies recognise multiple epitopes on a specific antigen Monoclonal antibodies recognise 1 epitope on a specific antigen
35
What is western blotting used for?
Using antibodies to detect a protein
36
How does western blotting work?
SDS-PAGE used --> addition of primary antibody which binds to the protein --> addition and binding of radioactively labelled secondary antibody ---> detection of protein
37
What is ELISA used to measure?
Measures the concentration of proteins in a solution
38
How does ELISA work?
Similar to western blotting, enzyme linked antibody binds to the protein --> substrate added and rate of reaction measured which is proportional to concentration
39
What is radioimmunoassay?
Used to measure the concentration of a product | Uses radiolabelled primary antibody
40
What are enzyme assays used for?
To measure the activity of an enzyme in solution through measuring of a product
41
What are two methods of continuous assays?
Spectrophotometry | Chemoluminescence
42
What are two methods of discontinuous assays?
Radioactivity | Chromatography
43
What can be used to measure the concentration of metabolites?
Enzymes
44
What is dsDNA and ssDNA?
Double stranded DNA Single stranded DNA
45
How can dsDNA be used to form ssDNA?
Denaturing - breaking of hydrogen bonds with heat or a pH>7
46
How can dsDNA be formed from ssDNA?
Renaturing - hydrogen bonds reformed | Cooling or pH=7
47
How can a DNA probe be used?
Denaturing of dsDNA into ssDNA ---> addition of complementary ssDNA sequence which is radioactively labelled ---> identification using photographic film
48
What are two hybridisation techniques?
Southern blotting | Northern blotting
49
What is southern blotting?
Uses DNA probes to identify complementary sequences after gel electrophoresis
50
What is northern blotting?
Similar to southern blotting, where DNA is used to detect RNA species
51
Is western blotting a hybridisation technique?
No - is the detection of proteins by antibodies after protein gel electrophoresis or SDS-PAGE
52
What is the process of southern blotting?
``` Digest DNA with restriction enzymes Separate with DNA gel electrophoresis Transfer dna fragments to nylon or nitrocellulose to denature Add radioactively labelled DNA probe Detection using X-ray film ```
53
Why is southern blotting used?
Used to detect dna fragments from complex mixtures
54
What are some characteristics of DNA probes?
DNA probes do not have to be 100% identical to the target sequence - will stand bind but less tightly DNA probes do not have to completely align with the target sequence
55
By which method is DNA sequencing carried out?
Sanger Chain Termination Method
56
What is a ddNTP?
Dideoxynucleotide triphosphate | Same as a normal deoxynucleotide triphosphate ---> but has a H atom on the 3rd carbon rather than a hydroxyl group
57
How are ddNTPs incorporated into a growing DNA strand?
Can be incorporated by DNA polymerase but will prevent further elongation of the chain as no further phosphodiester bonds can be formed
58
How is sanger chain DNA sequencing carried out?
Labelled primer + all the dNTPs + 1 ddNTP + DNA polymerase all placed in a test tube Different length DNA fragments produced depending on where ddNTP incorporated 4 separate test tubes used, DNA fragments separated by size on a gel Gel is read from the bottom to work out the nucleotide sequence
59
What techniques are used to investigate DNA at a chromosome level?
Karyotyping FISH Chromosome Painting
60
What techniques are used to investigate DNA at a nucleotide level?
DNA sequencing | PCR + restriction analysis
61
What techniques are used to investigate DNA at a gene level?
``` Southern hybridisation Northern hybridisation RT-PCR Microarray DNA fingerprinting ```
62
How can PCR be used to identify mutations?
By using allele specific primers
63
How does northern hybridisation differ to southern hybridisation?
Uses DNA probes to look at RNA species and investigate gene expression rather than gene structure
64
What is RT-PCR used for?
Used to look at gene expression
65
How does RT-PCR work?
A primer is added with a long stretch of T's at the 5' end to a mature mRNA molecule with a polyA tail Complementary sequence extended using reverse transcriptase RNA broken down with RNAases Forward and reverse primers added --> normal PCR reaction
66
What is microarray used to investigate?
To compare gene expression between two different sets of genes Allows the analysis of 1000s genes at the same time
67
What is DNA fingerprinting used to investigate?
To look for family relationships
68
How is dna fingerprinting used to look for family relationships?
There are repeating sequences on the non-coding part of DNA called minisatellites - these are unique to each individual and are inherited from mother and father --> so no individual will have the same Similarities between these sequences can be used to prove family relation
69
What is karyotyping? How can it be used to analyse chromosomes?
The extraction and staining of chromosomes at the metaphase stage Specific sizes/banding allows sorting and comparison
70
What is FISH? How is it used?
Fluorescent in situ hybridisation - happens inside the cell DNA probe with fluorescent dye added to cell Denaturing and hybridisation of DNA Detection of dna probe and its position on the chromosome take place

MCBG (15 decks)

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