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My MCBG > Molecular Techniques > Flashcards

Molecular Techniques Flashcards

1
Q

What is restriction analysis?

A

When specific endonucleases (restriction enzymes) recognise and cut out specific DNA sequences which can then be analysed (electrophoresis)

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2
Q

What is the region called which the Restriction enzyme recognises and what is special about it?

A

Restriciton sites
Palindromic (read same way forwards and backwards

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3
Q

Significance of restriction sites being Palindromic

A

Both DNA strands can have the DNA cleaved out

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4
Q

What is DNA Electrophoresis?

A

When an electrical current is used to separate DNA fragments based on their side through gel

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5
Q

How is DNA Electrophoresis set up?

A

Negative electrode on side of gel with wells that have the DNA deposited in them
Positive electrode opposite the wells

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6
Q

How does DNA Electrophoresis work?

A

DNA = NEGATIVELY CHARGED
DNA deposited in wells near negative electrode
Power supply turned on to generate charge difference across gel
DNA moves towards positive electrode
Smaller fragments move quicker so will move further up gel to +
Larger fragments move slower so further from +

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7
Q

So what 4 things are required for Gel Electrophoresis to work?

A

Gel (a matrix that allows DNA to separate)
Buffer (allows charge to be carried through Gel without pH changing)
Power supply (generate charge)
Stain/detection (to see DNA fragments)

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8
Q

Use of restriction analysis/ DNA electrophoresis

A

Investigate size of DNA fragments (deletions)
Investigate mutations (sickle cell changes DNA length)
DNA fingerprinting (Investigates DNA Variation)

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9
Q

What’s is a plasmid?

A

Small circular double stranded DNA
They carry genes to replicate independently and transfer to other bacteria

OFTEN carry ANTIBIOTIC RESISTANCE GENE (very useful in isolating bacteria which have taken up the recombinant plasmid)

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10
Q

Gene cloning process:

A

Isolate/remove gene with restriciton enzymes (creates sticky ends on gene)
Insert gene into Plasmid vector which also contains antibiotic resistance gene
Insert recombinant DNA into host like E.coli
Identify and isolate clone contains recombinant DNA/desired gene

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11
Q

What is recombinant DNA?

A

It is DNA that contains DNA from at least 2 different sources

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12
Q

How can we identify the clones that have taken up the desired gene in gene cloning?

A

Utilise the antibiotic resistance gene in the recombinant DNA
Expose all the bacteria to the Antibiotic
Bacteria that survive have desired gene

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13
Q

Polymerase Chain Reaction Function

A

To AMPLIFY a segment of DNA of interest

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14
Q

Key Enzyme involved in PCR

A

Taq polymerase

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15
Q

What is taq polymerase?

A

A DNA polymerase enzyme that replicates DNA at high temperatures

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16
Q

What is a PCR Primer

A

Short sequence of single stranded DNA that binds to and therefore marks the start and end of the section of DNA that will be amplified

Forward Primer binds to Start of 5’ strand of DNA
Reverse primer binds to start of 3’ strand of DNA

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17
Q

What does Anneal mean?

A

When temperature is lowered and PCR primer can bind to the start and end of DNA strand

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18
Q

PCR Process

A

Heat DNA 95C to denature(Separates 2 strands)
PCR Primer added (Both forward and Reverse Primer)
Cool it down so PCR Primers can anneal to the 2 strands
Taq polymerase can now start replicating the DNA from the primers
This is done many times amplifying the DNA exponentially

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19
Q

PCR BASIC process

A

Denature (Heat 95C)
Anneal (cool to add PCR Primers)
Replicate (taq polymerase)
Repeat

20
Q

How is PCR reaction important in genetic testing?

A

It produces many copies of a DNA segment which can then be used in many different analytical techniques

21
Q

Protein Gel Electrophoresis Function

A

Separates proteins based on size, charge or shape

22
Q

Protein Gel Electrophoresis process

A

Protein put in wells near negative electrode
Current causes the charged protein molecules to separate, the negative charges wil go more towards the positive electrode

23
Q

Protein Gel Electrophoresis 4 requirements (same as DNA electrophoresis)

A

Gel
Buffer
Power supply
Stain/detection

24
Q

What is an antigen?

A

The specific protein target region that an antibody binds to

25
Q

What is an epitope?

A

It is a sequence of amino acids within the antigen that the antibody recognises

26
Q

2 types of Antibody:

A

Monoclonal Antibodies
Polyclonal Antibodies

27
Q

About Monoclonal Antibodies

A

Made by 1 B lymphocyte
All identical (1 type) antibody
Binds to 1 antigen
Only 1 Epitope

28
Q

Polyclonal antibodies

A

Made by many different B lymphocytes
Different antibodies
Different antibodies recognise different epitopes
1 specific antigen

29
Q

Basic description of how polyclonal antibodies work

A

The different antibodies all bind to the one antigen however they bind to this antigen at different points, these different points are called epitopes

30
Q

Antibodies can label/detect proteins

A

Primary antibody binds to antigen
Secondary antibody has fluorescent tag bound
Secondary antibody binds to primary antibody

31
Q

Western Blotting to detect proteins

A

Protein electrophoresis separates out the Proteins
Primary Antibodies (PA) added to proteins
Unbound PA washed away
Secondary Antibodies (SA) added and bind to PA
Unbound SA washed away
The SA have an enzyme attached
Substrate added, enzyme on SA converts substrate producing illumine-scent signal

32
Q

Enzyme Assay Function

A

Used to measure rate of chemical reaction in different samples

33
Q

Enzyme linked immunosorbent assay (ELISA) Function

A

Used to measure concentration of proteins in a solution

The faster the colour change the higher the conc

34
Q

2 Types of Enzyme assay

A

Continuos
Discontinuous

35
Q

Continuous enzyme assays

A

Give real time read of protein level
Spectrophotometer
Chemluminescence

36
Q

Discontinuous enzyme assay

A

Gives snap shot of protein level at one point in time

Radioactivity
Chromatography

37
Q

Enzyme assay uses

A

Diagnosis of diseases
Either metabolic disorders (test for protein)
Diagnose disease by detecting presence or lack of serum enzymes

38
Q

Important serum Enzymes that Enzyme assays can test for in diseases

A

Liver disease/damage = Aspartate Transaminase (AST), Alanine Transaminase (ALT)
Marker for liver damage (alcoholism) = g-glutamyltransferase

Bone disorders = Alkaline phosphatase

39
Q

Enzyme assays can be used to detect acute myocardial infarction

A

Levels of Cardiac Troponin increase after
Creatin Kinase levels also increases

40
Q

What direction d forward primers read?

A

From 5’ prime to 3’ prime

41
Q

What direction do reverse primers read?

A

3’ prime to 5’ prime

42
Q

What causes Sickle Cell Disease?

A

Single base mutation
A ———> T
Purine ——-> Pyrimidine. = Transversion substitution

43
Q

What gene is mutated in Sickle Cell Disease?

A

HBB Gene
Codes for Beta Globin Chains in haemoglobin

44
Q

How does the mutation of A —-> T in Sickle Cell cause issues?

A

Destroys an MstII site in the HBB gene
So unhealthy allele not cut into 2 by restriction enzymes

In Sickle cell alleles, no restriction site means restriction enzymes don’t cut up DNA fragment so only 1 fragment per allele

45
Q

Serum Protein Electrophoresis detects globulins in the blood. ITs uses are too?

A

Used to diagnose diseases:
- Multiple myeloma
-Anaemia and Chronic Kidney Disease

My MCBG (2 decks)

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