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Sem 1 - MCBG > Analysis Techniques > Flashcards

Analysis Techniques Flashcards

1
Q

Wha happens when double stranded DNA is heated or treated with alkaline solution?

A

It denatures which breaks the hydrogen bonds and releases single stranded DNA (ssDNA)

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2
Q

What is the basis for DNA hybridisation?

A
  • dsDNA is heated so that the hydrogen bonds are broken and it is denatured.
  • Then, before cooling, complimentary ssDNA with a radioactive or fluorescent marker is added.
  • some of the original ssDNA will anneal with the labelled DNA
  • This labelled DNA can now be identified using photographic film.
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3
Q

What are the differences between Southern, northern and western blotting?

A
  • Southern blotting was invented by (and named after) after Prof Sir Ed Southern in 1975 and to uses probes to identify complimentary DNA sequences after gel electrophoresis.
  • Northern blotting was invented in the late 1970s (not named after a person) and it used DNA to detect RNA species in a similar way to southern blotting.
  • Western blotting is NOT a DNA hybridisation technique. It uses similar principles to detect proteins using antibodies after protein gel electrophoresis.
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4
Q

What are the stages of Southern blotting?

A
  1. Digest DNA using restriction enzymes,
  2. Use DNA gel electrophoresis to separate the fragments of DNA,
  3. Transfer DNA fragments to a nylon or nitrocellulose membrane and soaked in alkali to denature the DNA,
  4. Hybridise filter with a labelled gene probe to detect a specific piece of DNA,
  5. Wash the filter to remove any unbound probe and detect hybridisation (and therefore DNA of interest) by exposing the filter to an X-Ray film.
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5
Q

Why do we use Southern blotting?

A
  • To investigate gene structure. Eg large deletions of duplications
  • To investigate gene explanation such as triplet repeats. Eg Fragile X syndrome and Huntington’s,
  • To investigate mutations in genetic tests - this uses allele specific probes eg for Sickle Cell disease,
  • To investigate variation and genetic relationships eg DNA fingerprinting.
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6
Q

What other technique is Southern blotting often used in combination with and why?

A

PCR to allow easier identification as PCR amplifies the gene. This means that Southern blotting allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.

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7
Q

What two things do probes not have to be?

A

Probes do not have to have 100% similarity to the target sequence. 80% would be enough. All this means is that it would bind less tightly.
Probes also do not have to completely align with the target sequence. Even if it only binds to part of the sequence, it is enough to allow detection.

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8
Q

What do probes not affect?

A

Probes do not affect the position of a target sequence on a gel. This is because the number of base pairs and therefore length of the sequence will be the same.

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9
Q

What is the purpose of the Sanger chain method?

A

It is also known as the dideoxy chain termination method. It allows us to work out the nucleotide sequence of DNA depending on when termination occurs.

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10
Q

What is a ddNTP

A

A ddNTP is a dideoxynucleotide triphosphate. It is the same as a normal DNTP (base) which is usually used in DNA replication except, it has a H rather than an OH at 3’. This means that, if this binds, DNA replication cannot continue because there is no OH to form the phosphodiester bond with the next base.

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11
Q

What is the Sanger chain termination method?

A
  • In this method, 4 separate test tubes are used. Each with a different ddNTP in (A,C,T,G), the DNA and primers to initiate DNA replication.
  • These test tubes are incubated at 37°C and DNA left to replicate.
  • After incubation, the products of the reaction are run out using separate lanes for each of the reaction test tubes. This will separate the labelled fragments out on the basis of size.
  • We are then able to read off the sequence from the bottom of the gel to work out the nucleotide sequence in a newly synthesised strand.
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12
Q

What variation on the Sanger chain termination method is used now?

A

Now, we use fluorescently labelled ddNTPs all in the same tube. The different length fragments are then separated on a very thin capillary. As the fragments fall off the end, they are detected by a laser. This is then produced as a chromatogram where read the sequence directly.

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13
Q

What restriction endonucleases? What are they used for?

A

They are usually very specific palindromic sequences (same forwards as backwards) produced by bacteria. They:

  • Recognise and degrade foreign DNA
  • Recognise and cut specific DNA sequences
  • They are ‘MOLECULAR SCISSORS’
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14
Q

What are the four requirements for DNA gel electrophoresis?

A
  1. Gel -A matrix that allows for the separation of DNA fragments
  2. Buffer- Allows charge on the DNA samples across the gel
  3. Power Supply - This generates a charge difference across the cell
  4. Stain/Detection -eg ethidium bromide to allow the identification of the presence of the separated DNA.
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15
Q

Why can gel electrophoresis be used to separate DNA molecules of different sizes?

A

Because DNA is negatively charged (the phosphate) and so will move towards the positive electrode (the anode) is placed in an electric field. The larger fragments move slowly and smaller fragments move faster which then allows the separation to occur.

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16
Q

Why do we use restriction analysis?

A
  • To investigate the size of DNA fragments eg small deletions
  • To investigate mutations eg sickle cell disease
  • To investigate DNA variation eg DNA fingerprinting
  • To clone DNA
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17
Q

What are plasmids?

A

They are small, circular dsDNA that are found in bacteria. The are like “mini chromosomes” as they carry genes to replicate independently. They also often carry antibiotic resistant genes and can be transferred to other bacteria.

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18
Q

What are the four basic steps of gene cloning?

A
  1. Isolate the relevant gene of interest following PCR and digestion with restriction enzymes
  2. Insert gene of interest into a plasmid vector (to create a recombinant DNA molecule)
  3. Introduce the recombinant DNA molecule into suitable host cells eg E. coli
  4. Identify and isolate the clone containing the gene of interest.
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19
Q

Why do we clone human genes?

A

To make useful proteins eg insulin
To find out what genes do or the difference between individuals
Genetic screening eg for Huntington’s, BRACA1/2, Cystic Fibrosis
Gene therapy eg cystic fibrosis (get wild type (normal) gene into patient using inhalers)

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20
Q

What is PCR?

A

PCR is the polymerase chain reaction and it is used to amplify target DNA.

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21
Q

Describe the process of PCR

A
  • Heat the DNA to 95°C to DENATURE it (break the H bonds).
  • Cool to 60°C to allow both the specific forwards and reverse primers (sequence of Oligonucleotides) to ANNEAL to the DNA strands.
  • Heat to 72°C which is the optimum temperature for Taq Polymerase to add new nucleotides to the strand (POLYMERISE).
  • Repeat 20-35 times. This results in an exponential increase in DNA.
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22
Q

Why do we use PCR?

A

To amplify a specific DNA fragment
To investigate specific single base mutations eg Tay Sachs and Sickle Cell
To investigate small deletions or insertions eg Cystic Fibrosis
To investigate variation and genetic relationships eg DNA profiling or DNA typing or DNA fingerprinting.

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23
Q

What are the differences between protein and DNA gel electrophoresis?

A
  • Proteins can be either positive or negatively charged so can move towards the anode or the cathode. DNA always moves towards the anode as it is negatively charged.
  • Protein electrophoresis is conducted vertically whereas the DNA electrophoresis is conducted horizontally.
  • In protein, it is quantitative so you can different intensities of colour or peaks on a graph to show the level of protein (eg in blood serum).
  • Protein electrophoresis is most commonly used to diagnose multiple myeloma
24
Q

What is isoelectric focusing?

A

IEF is when proteins separate on the basis of charge. Proteins migrate in a gel with a stable pH gradient (created using an electric field) until they reach a neutral pH equal to their pI. They they reach this point, there is no net charge so they stop migrating.

25
Q

What is SDS-PAGE?

A

This is when proteins separate according to their size.
SDS is used to denature the proteins, then it is run though electrophoresis (PAGE). Because the charges are almost identical in the denatured proteins, the differences in distance travelled will be because of size only. An unknown can be run alongside known values to work out the length.

26
Q

What is 2D-PAGE?

A

This is two dimensional electrophoresis. This allows the separation of complex mixtures of proteins because it separates then according to charge and then size. This technique is important for diagnosing diseased states in different tissues.

27
Q

How do you identify proteins?

A
  • Digest Protein with trypsin
  • perform mass spectrometry
  • Generate a list of peptide sizes
  • Use the database of predicted peptide size for known proteins to identify the protein
28
Q

What are polyclonal antibodies?

A

These are produced by many B lymphocytes so are many different antibodies specific to one antigen. There are multiple epitopes.

29
Q

What is an epitope?

A

An epitope is also known as antigenic determinant. It is the part of of an antigen that is recognised by the immune system, specifically B and T cells.

30
Q

What are monoclonal antibodies?

A

There are produced from one B lymphocyte so it is only one identical antibody that is specific to one antigen. Therefore, there is only one epitope.

31
Q

How is western blotting used to detect proteins?

A
  1. Protein is boiled so that it is denatured (to ensure separation according to size)
  2. Proteins are separated using electrophoresis (SDS-PAGE)
  3. Proteins are transferred to a nitrocellulose or PVDF membrane
  4. Blocking agent then used to prevent non specific antibody binding
  5. Stained with antibodies specific to the target protein
  6. Membrane stained with secondary antibody that recognises the first antibody staining. This is used for detection using a variety of techniques.
32
Q

Describe the steps of an indirect ELISA

A
  1. Antigen coated well
  2. WASH
  3. Specific antibodies bind to the antigen
  4. WASH
  5. Enzyme linked antibody bind to the specific antibody
  6. WASH
  7. Substrate is added and converted by the enzyme into a coloured product. The rate of colour formation is proportional to the amount of specific antibody.
33
Q

What other technique works in the same as as ELISA?

A

Radioimmunoassay. But, it used a radiolabelled primary antibody.

34
Q

What can an indirect ELISA be used for?

A

It can be used to measure the concentration of of proteins in solution eg Hormones - Insulin, cortisol, TSH.

35
Q

What do Enzyme assays do?

A

They measure the rate of product production / rate of enzyme activity over time.

36
Q

Give two examples of continuous assays

A
  • Spectrophotometry

- Chemoluminescence

37
Q

Give two examples of discontinuous assays

A
  • Radioactivity

- Chromatography

38
Q

What can the measurement of enzymes tell us?

A

The enzymes in tissues can tell us about metabolic disorders and serum enzymes can help with the diagnosis of disease. Eg if an enzyme is in serum and it shouldn’t be then it is a marker for damaged tissue and therefore disease.

39
Q

What is the gold standard measurement for the diagnosis of a myocardial infarction?

A

The measurement of cardiac troponin I (cTnI) by ELIZA.

CK-1 is also raised in the brain and CK-2 is raised in the myocardium and CK-3 in the the skeletal muscle and myocardium.

40
Q

What can you do after PCR?

A
  • Restriction analysis
  • Gel Electrophoresis
  • DNA sequencing
  • Southern Blotting
  • Another PCR (‘nested PCR)
41
Q

Where are restriction enzymes found?

A

Bacteria

42
Q

Why was PCR on RNA negative?

A

Because Tap Polymerase could not bind (as it is a DNA polymerase)

43
Q

Who would be interested in accessing your genome information?

A 
Family 
Potential spouse 
Doctors 
Government 
Police 
Schools 
Insurance companies (could lead to discrimination...)
44
Q

Who owns your DNA sequence?

A 
You?
Sequencer?
Person who paid?
Parents? 
Anybody?

This is an ethical issue if DNA sequencing is done regularly.

45
Q

When would direct to consumer genetic testing be used?

A

Paternity testing
Presence of a particular gene - sports gene, Health eg cancer susceptibility?
Gene partner / matchmaking

46
Q

When looking at primers for PCR, what end 5’ or 3’, is it more important to be perfectly complimentary to the template strand?

A

It is more important that the 3’ is copied accurate because this is the end that gets elongated. If it is not, then DNA replication will not occur. This is called using “allele specific primers”

47
Q

In what technique and why are allele specific probes useful?

A

Allele specific probes are used in Southern hybridisation. At normal conditions, both the specific and slightly mutated probe will bind. However, if you change the conditions to make them more extreme, only the specific probe will bind. This can be used to see if an individual has a specific gene eg sickle cell.

48
Q

What is rt-PCR?

A

Reverse transcriptase PCR. This is when you replicate mRNA.

49
Q

Describe how the process of reserve transcriptase PCR works?

A

This technique is used to amplify mRNA.
First, you use reverse transcriptase to convert mRNA to cDNA (copy DNA) . This cDNA will have lots of Ts at the start which compliment the poly A tail of the RNA so, the primer will have lots of Ts.
Next, add RNAase to degrade the RNA and not the DNA. You are now left with single stranded DNA.
After this, you design a forward and reverse primer. The forward primer will make the DNA double stranded and you now have amplified mRNA (as DNA)

50
Q

Why is reverse transcriptase PCR useful?

A

To look at gene expression as will have mRNA.

51
Q

What is microarray technology used for?

A

Microarray technology is used for genome wide analysis. It is used to compare two conditions eg healthy and cancer.

52
Q

How does microarray work when comparing two conditions?

A

Two petri dishes for different conditions.
Use reverse transcriptase to make fluorescently labelled cDNA from mRNA. Different fluorescent labelled for each condition.
This creates two pools of DNA with different labels.
Mix both and hybridise with an array containing all the human genome.
This will show what genes are expressed in what cells. Eg if expressed in both, it will be a mix of both colours whereas, if only one colour, this gene is only switched on in this type of cell.

Powerful method as can look at all genes at the same time.
Looks at conditional gene expression.
Or at mutations eg micro duplication or micro deletions in patients with a condition (array comparative genome hybridisation).

53
Q

How does DNA fingerprinting Work?

A

It was found out that different people have a different number of multiple repeats at different loci in the chromosomes. These multiple repeats can be cut using restriction enzymes and then they undergo DNA hybridisation. This creates a unique pattern for each individual and can be used to show inheritance patterns.

54
Q

What is DNA profiling?

A

Every lane becomes into peaks with numbers. The numbers ate stored in a data bank when arrested and they relate to the amount of repeats at highly variable regions. This is how DNA profiling works. Same principle as DNA fingerprinting.

55
Q

What is Karyotyping?

A

This is analysis of DNA as the chromosome level. You create a metaphase spread and look at the banding patterns of each of the chromosomes to work out what chromosomes they are (eg if 3x chromosome 21 it’s Down syndrome) or if banding patterns are abnormal.

56
Q

What is FISH?

A

Fluorescent in situ hybridisation. We make probes and look at where they bind in cells.
Eg used for Williams syndrome as know the region where mutation occurs.
Eg used for chromosome painting. Use same fluorescent tag for genes on a particular chromosome to make all chromosomes different colours. This makes them easily identifiable.

Sem 1 - MCBG (16 decks)

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