Molecular Techniques and Diagnosis Flashcards
What produces endonucleases?
Bacteria
What do endonucleases do?
Recognition and degradation of foreign DNA
What are specific endonucleases also known as?
Restriction enzymes
What do specific endonucleases do?
Cleave phosphodiester bonds inside DNA
What is the specific place recognised and cut by a restriction enzyme called?
Restriction site
What is usually formed on cutting with a restriction enzyme?
- (Mostly) palindromes of 4,5,6,8 bp
- (Mostly) staggered cuts, leaving ends
What is special about palindromes?
They read the same in the forward and backwards direction
What happens if a cut leaves sticky ends?
If they come back together, they will be held together by hydrogen bonds
What can be produced in contrast to sticky ends?
Blunt ends
How is DNA protected?
Methlation
What happens in methylation?
Methyl groups are added on my some enzymes
What can restriction enzymes be used to do?
Isolate DNA fragments
What is the purpose of DNA gel electrophoresis?
Separates DNA fragments produced
What happens in DNA gel electrophoresis?
- DNA samples loaded in wells
- Moves along agarose
- DNA fragments move from -ve electrode to +ve electrode
What is the purpose of the agarose in DNA gel electrophoresis?
Acts as molecular sieve
Why do DNA fragments move from the -ve to the +ve electrode?
As DNA is -vely charged
What fragments move furthest in DNA gel electrophoresis?
The smallest
What are DNA fragments separated on the basis of in DNA gel electrophoresis?
NAME?
What is required for gel electrophoresis?
- Gel
- Buffer
- Power supply
- Stain/detection
What is the gel in gel electrophoresis?
A matrix that allows for separation of DNA fragments
What is the purpose of the buffer in gel electrophoresis?
Allows charge on DNA samples across gel
What is the buffer in gel electrophoresis?
A liquid containing salt
What does the power supply in gel electrophoresis do?
Generates charge difference across the gel
What is the purpose of the stain in gel electrophoresis?
To identify the presence of the separated DNA
What is often the stain in gel electrophoresis?
A molecule that sits between bases of DNA that fluoresces when light shines
What are the uses of restriction analysis?
- To investigate the size of DNA fragments
- To investigate mutations
- To investigate DNA variation
- To clone DNA
What is DNA ligase?
A ‘molecular glue’
What does DNA ligase do?
Makes new phosphodiester bonds
What is DNA ligase used for?
To join DNA of interest to DNA of vector
How can DNA ligase be used to join DNA of interest to DNA of vector?
DNA fragments cut with the same restriction endonuclease has the same overhanging fragments, and so can be mixed with DNA ligase
How can the same effect as cutting two pieces of DNA with the same restriction endonucleases be achieved?
By using different restriction enzymes giving the same complementary sequences
What are plasmids?
- Small circular dsDNA
- ‘Mini-chromosomes’
Where are plasmids found?
In bacteria
What do plasmids do?
NAME?
What do plasmids have the ability to do?
Transfer to other bacteria
What do plasmids often carry?
Antibiotic resistance genes
What happens in the process of gene cloning?
- DNA of interest is joined with a plasmid vector, forming a recombinant DNA molecule
- Recombinant DNA molecule is introduced into bacterium, where replication will take place
- Cells containing recombinant DNA are selected, identified and isolated
What is the process of introducing the recombinant DNA molecule into the bacterium called?
Transformation
Why clone human genes?
- To make useful proteins
- To find out what genes do
- Genetic screening
- Gene therapy
How is mammalian proinsulin mRNA obtained?
From the pancreas
How is proinsulin cDNA produced?
From mammalian proinsulin, by action of reverse transcriptase
How is cDNA cloned?
- cDNA joined to plasmid, forming recombinant plasmid
- Recombinant plasmid infects E.coli, producing transformed bacteria
How is the PCR carried out?
- DNA molecule heated to 95º c , denaturing it
- Primers added
- DNA cooled to RT, so renatures with primers attached
- Add DNA polymerase
Why does heating the DNA molecule denature it?
The hydrogen bonds holding it together break
What do the primers in PCR do?
Define the region to be amplified
How does DNA polymerase extend the DNA molecule?
5’ to 3’, starting at primers
Why is thermostable DNA polymerase (Taq) used in PCR?
Because it means we don’t have to add new enzyme every time
Why is a pair of primers used in PCR?
The forward and reverse
What do the primers in PCR do?
Uniquely define the region to be copied
What does the temperature achieve in PCR?
Cycles of denaturing, annealing, and polymerising
What is used to cycle temperatures in PCR?
Thermocycler
What does repeated PCR result in?
Exponential increase in DNA
Why use PCR?
- To amplify a specific DNA fragment
- To investigate single base mutations
- To investigate small deletions or insertions
- To investigate variation and genetic relationships
What happens to proteins if placed in electric field?
They will move towards the anode or cathode
Why will proteins move towards anode or cathode in electric field?
They are charged molecules
What are proteins separated on the basis of?
NAME?
What are the requirements for gel electrophoresis?
The same as for DNA
What is the process for carrying out protein gel electrophoresis?
NAME?
In protein gel electrophoresis, what charge goes on what end?
Doesn’t matter, because proteins have different charge if looking at native proteins
What does protein gel electrophoresis produce?
Bands
What do darkly stained bands in protein gel electrophoresis indicate?
More protein present
What does SDS-PAGE separate proteins on the basis of?
Size
What does SDS-PAGE rely on using?
Unfolded proteins
Why does SDS-PAGE rely on using unfolded proteins?
Because the shape of protein affects how it moves in a gel, and it has an intrinsic charge
What is used to denature proteins in SDS-PAGE?
- ß-Me
- SDS
What does ß-Me do?
Breaks disulphide bonds
What is the purpose of the addition of ß-Me and SDS in SDS-PAGE?
It removes the tertiary and secondary structure, to give a linear polypeptide change
What does SDS do?
Binds in a fixed way to amino acid sequence
What has happened after the addition of the agents in SDS-PAGE?
It now has a uniform negative charge, so can be separated by gel electrophoresis
What does SDS-PAGE allow for?
Analysis of proteins
What can be do if we know the size of a particular band produced by SDS-PAGE?
Go looking for it in a mixture of proteins
What can be looked for using SDS-PAGE?
- Protein expression
- Levels of protein
- Protein presence
What basis are proteins separated on in isoelectric focusing (IEF)?
Purely charge
Describe the method for IEF
- A stable pH gradient is established in the gel after application of an electric field
- Protein solution added, and electric field reapplied
- After staining, proteins shown to be distributed along pH gradient according to their pI values
Why does IEF work?
Because proteins migrate until they reach a pH equal to their pI. There is no net charge at pI, so they stop migrating
What does 2D-PAGE make use of?
IEF, followed by SDS-PAGE
What does 2D-PAGE separate based on?
Size and charge
What does 2D-PAGE allow for?
The separation of complex mixture of proteins
Where is 2D-PAGE important?
For diagnosing disease states in different tissues
How can 2D-PAGE be used to diagnose?
Can look at changes by comparing normal conditions to disease conditions
What is protein identification also known as?
Proteomics
How can proteins be identified?
Can take proteins from gel, and identify using mass spectrometry, using a database
Describe the method for proteomics
- Digest protein with trypsin
- Perform mass spectrometry
- Generate list of peptide sizes
- Uses database of predicted peptide sizes for known proteins to identify protein
How can specific cleavage of proteins be carried out?
- Enzymatic cleavage
- Chemical cleavage
What happens in enzymatic cleavage?
Enzymes break peptide bonds in particular protein sequences
Give 2 examples of enzymes used in enzymatic cleavage
- Trypsin
- Staphylococcal protease
Give 2 examples of chemicals used in chemical cleavage?
- Cyanogen bromide
- Hydroxylamine
What is proteomics?
Analysis of all proteins expressed from a genome
What is molecular diagnosis?
Analysis of a single purified protein
What do antibodies do?
Bind to a specific protein targets called antigens
What do antibodies recognise?
Epitopes
What is an epitope?
A few amino acids on a protein
What produces polyclonal antibodies?
Many B lymphocytes
What are polyclonal antibodies?
Multiple different antibodies
What are polyclonal antibodies specific to?
1 antigen
How many epitopes do polyclonal antibodies have?
Multiple
What must be done to obtain polyclonal antibodies?
You inject with the antigen 3-4 times at two week intervals, and then bleed. You can then get isolated antibodies from extracted blood
What produces monoclonal antibodies?
1 B lymphocyte
What are monoclonal antibodies?
1 identical antibody
How many epitopes do monoclonal antibodies have?
1
How to you obtain monoclonal antibodies?
- Inject with antigen
- Obtain spleen cells
- Mix spleen cells with myeloma cells. Culture the myeloma line, and then fuse in polyethylene glycol
- Select and grow hybrid cells
- Freeze and thaw the cells as required
- Can inject into mice to induce tumours, extracting the antibody this way, or grow the hybrid cells in mass culture and then extract the antibodies they produce
What is the purpose of fusing the spleen cells with myeloma cells?
Gives immortal cell life, as they just keep dividing
What hybrid cells do you select for when producing monoclonal antibodies?
The ones making antibodies of desired specificity
What does the western blotting process involve?
Detection of proteins on SDS-PAGE gel by antibodies
Describe the process of western blotting
NAME?
What solid matrix is the SDS-PAGE usually transferred onto?
Usually nitrocellulose
What does the transference of the SDS-PAGE onto a more solid matrix produce?
A replica of gel electrophoretogram that is a more permanent record
What is important of the antibody used against the protein of interest?
It must be specific to the protein of interest
Is the protein of interest in western blotting folded or unfolded?
Doesn’t mattter
What must be true of the enzyme-linked second antibody?
It must have some sort of marker
What is the purpose of the enzyme-linked second antibody?
Detects the binding of the first antibody
What is produced from western blotting?
An immunoblot
How is an immunoblot interpreted?
By the band, which appears in a specific place
What can an immunoblot be used to determine?
- The expression of protein
- How much protein’s being produced
What can ELISA measure?
The concentration of a particular protein in solution
What can ELISA be used to work out?
How much antibody binds at a given antigen concentration
How can ELISA be used to work out how much antigen binds at a given antigen concentration?
By building up a standard curve
Describe the process of ELISA?
NAME?
What is the significance in the rate of colour formation in ELISA?
It is proportional to the amount of specific antibody
How does radioimmunoassay work?
In the same way as ELISA, but uses radio labelled primary antibodies
What are enzyme assays used to do?
Work out normal activity
What can enzyme assays be used to do?
Compare against abnormal cases
What do enzyme assays measure?
The amount of product at any given time
Are enzyme assays continuous or discontinuous?
Can be either
Give 2 examples of continuous assays
- Spectrophotometry
- Chemiluminescence
Give 2 examples of discontinuous assays?
- Radioactivity
- Chromatography
Where may in be clinically important to measure enzymes?
NAME?
What can measurement of enzymes in tissues detect?
Metabolic disorders
What can measurement of serum enzymes do?
Diagnose disease
Give 2 examples of clinically important serum enzymes
NAME?
When can some important serum enzymes indicate disease?
When they are found in the serum, as they are not normally
Give an important condition that abnormal presence of serum enzyme indicates?
A heart attack
What is the gold standard diagnosis method of MI?
Measurement of cardiac troponin by ELISA
What can enzymes be used to measure?
Clinically important metabolites
Give an example of how enzymes can be used to measure clinically important metabolites?
Measurement of glucose conc. with glucose oxidase
Where is measurement of glucose conc. using glucose oxidase used?
In biosensor of glucose
What is required for the Sanger chain termination method?
NAME?
How do the labelled nucleotides terminate polypeptide production?
They are dideoxy on C3 and C2
What happens in the Sanger chain termination method?
The normal and labelled nucleotides are mixed together, producing coloured bands
What is the significance of the bands produced in the Sanger termination method?
Each band is one nucleotide difference
How is the Sanger chain termination method interpreted?
The computer reads it, detects the colour of the band and uses this to sequence the DNA
Why has the cost of sequencing fallen?
Due to advancement of next generation sequencing
What is the consequence of the falling cost of sequencing?
Becomes viable to get a genome sequence done
What are the ethical considerations regarding DNA sequencing?
- Who has access to the genome sequence
- Can knowledge help prevent illness later in life
- Does it open up areas for discrimination
- Who owns the DNA sequence
Who may be interested in knowing a persons genome information?
- Family
- Potential spouse
- Doctors
- Government
- Police
- Schools
- Insurance companies
How is DNA hybridisation using a probe carried out?
- Denature the DNA
- Make a probe that is complementary to the DNA sequence, and add a fluorescent or radioactive label
- Renature the DNA. Some of the DNA will bind to the probes
What is formed when DNA binds to a probe?
A heteroduplex
What is the result of the formation of a heteroduplex?
The DNA can be seen in a blot
What does Southern hybridisation use?
An allele-specific probe
How can probes be made very specific?
Using oligonucleotides
How can an probe using oligonucleotides detect disease?
A normal oligonucleotide will bind when the sequence is normal, but won’t bind as tightly with a mutant allele, as there is a mismatch. Vice versa for disease specific oligonucleotides
What does PCR using allele specific primers mean
Only DNA with an allele complementary to the probe is detected, and so only amplify normal DNA (or mutant DNA)
What would happen if you attempted PCR using allele specific primers with a mismatch at the 5’ end?
It wouldn’t matter that much, and so PCR can continue
What would happen if you attempted PCR using allele specific primers with a mismatch at the 3’ end?
Very dramatic, as if the 3’ end was mismatched, Taq polymerase can’t bind as the template can’t be read
What does Northern hybridisation look at?
How much of a gene is expressed, by looking at how much RNA is made
What is the difference between Northern and Southern hybridisation?
RNA is used in Northern
Why must care be taken in Northern hybridisation?
RNA degrades
What happens in Northern hybridisation?
You make a gel with RNA, and separate the RNA fragments
What can be seen from Northern hybridisation?
NAME?
What can reverse transcriptase do?
Take copy of a single stranded mRNA, and make it into DNA
What do you have when a gene is expressed?
mRNA
What could you say if used mRNA to do PCR?
Could say when mRNA is made
What must be done to mRNA to perform PCR?
PCR can’t work with RNA, so need to make it into RNA
What does every mRNA have?
A long polyA tail
What is required to make mRNA into DNA?
A T primer, that binds to the polyA tail, so there is a template to copy
What type of DNA is formed from mRNA?
cDNA
What happens once cDNA is made?
The RNA is degraded using nucleases
What is used to degrade the RNA?
An enzyme that specifically degrades RNA
What can you do once you have cDNA produced from RNA?
Continue PCR
What do you need for PCR using cDNA?
Forward and reverse primers
Why do you need forward and reverse primers for PCR with cDNA?
Forward primer uses template to make another strand, producing a double strand. Once you have double strand, CPR continues as normal
What is microarray technology looking at?
Conditional expression, so what RNA is produced, and therefore what’s actually being expressed in healthy and tumour tissue
What must always be done in microarray technology?
Comparison of two conditions
Describe the process of microarray technology
- Have samples of both cells, and isolate the RNA
- Use reverse transcription labelling to make cDNA with coloured fluorescent probe
- Combine targets by mixing in equal quantities
- Hybridise to microarray- probes find complementary sequences on individual dots, and bind to it
What will happen if a gene is switched on in a tumour tissue, but not under normal conditions?
We will only see the colour of the tumour cDNA marker
When may we use array comparative genome hybridisation?
If we have a suspected chromosomal problem, but don’t know where in the genome is wrong
Describe the process of array comparative genome hybridisation
- Extract DNA from both cell types
- Label DNA with two different flourochromes
- Mix in equal quantities and hybridise with microarray slide
What must we know about the microarray slide?
Must know which genes are represented by which dot on the slide
What happens if there is nothing wrong at a certain position on the chromosome?
Equal colours
What happens if there is a deletion at a chromosomal location?
Bits of DNA that don’t have one of the colours
How can a disease be determined?
By working out red:green ratio
Why use array technology?
- To investigate 1000’s genes at the same time
- Investigate chromosome deletions/duplications
- Investigate conditional gene expressoin
Where is array technology better than Northern and Southern blotting?
When you need to look at many genes, or don’t know what genes you want to look at
What does array technology allow for?
Genome wide analysis
When will array technology be used to investigate chromosome deletions/duplications?
When micro deletions/substitutions
Why will array technology only used to investigate chromosome changes when they are small?
If they are bigger, can physically see them
What does DNA fingerprinting use?
Minisatellites
What are minisatellites?
DNA sequences that are repeated over and over again
Why are minisatellites used in DNA fingerprinting?
They show copy number variation, and everyone has different number of copies, so they are highly variable regions
What does DNA profiling use?
Small Tandem Repeats (STRs)
What are STRs?
Like minisatellites but only a few bases
How do we carry out DNA profiling?
Do PCR on one of the variable regions
Where can DNA profiling be used?
NAME?
What happens in Karyotyping?
Stain chromosomes, so can see banding patterns
Give an example of a problem that could be seen using Karyotyping?
A translocation
What is a translocation?
When bits of chromosomes have fused with other chromosomes
What happens in FISH?
- Label probe DNA with fluorescent dye
- Denature and hybridise with chromosomal DNA
What is required for FISH?
Need to know what you’re looking for
How does FISH work?
If you have a gene you suspect has something wrong with it, make probe DNA for that gene, and label inside a cultured cell. The probe will bind at the position the gene is in the chromosome, and give a fluorescent tag.
What does chromosome painting do?
Labels all chromosomes a different colour
What makes chromosome painting possible?
We know exactly whats on each chromosome, so can make probes for sequences of DNA on a chromosome, and label all probes from one chromosome in the same colour
What are the uses of FISH?
NAME?
