Molecules, Genes And Disease

Front 1

Describe gene cloning and restriction analysis

Back 1

Restriction anaylysis/ Gene cloning – restriction enzymes are endonucleases which bind to restriction sites in the template DNA and cut out specific DNA sequences- DNA fragments. Restriction sites are palindromic ( can be read in both ways) and only a few base pairs long. The double stranded break in DNA can be repaired by DNA ligase which allows genes to be ‘cut and paste’ leading to DNA modification. In gene cloning the DNA fragment is inserted into plasmid vector, by DNA ligase, forming recombinant DNA molecule. Plasmid vector is then transferred into bacterial cell by horizontal transmission and cell undergoes rapid division and the cells containing recombinant DNA are identified and isolated. A clone is a colony of identical cells containing the recombinant DNA. This is used to make useful proteins eg insulin, to find out what genes do eg HTT, genetic screening eg huntingtons and potential genetic therapy eg cystic fibrosis
Mutations may cause addition or removal of restriction sites and so use of restriction enzymes and DNA sequencing can identy the mutation.

Front 2

describe DNA sequencing.

Back 2

DNA sequencing is the process where the sequence of nucleotides in a DNA molecule is determined. Sanger dideoxy chain termination method is used where 4 different dideoxynucleotide tri phosphates that are radioactively labelled or flouresent are incorporated with the DNA template strand, d.nucleotides , DNA primer (initiates DNA synthesis) and DNA polymerase individually. The dideoxynucleotides terminate DNA elongation as they don’t have a 3’ OH group so phosphodiester bonds can’t form. The DNA molecules are denatured, to separate the strands and separated by size by DNA gel electrophoresis. The DNA bands are then visualised by autoradiography or UV light and seen in an Xray film.

Front 3

Describe the theory behind DNA electrophoresis and how technique can be used to provide information about DNA fragments.

Back 3

DNA molecules are negatively charged and so will move towards the anode if placed in an electric field. DNA fragments can be separated on the basis of size by this method, the smaller the fragment the faster it moves across the gel. An agarose gel is required that allows the separation of fragments, a buffer allows charge on the DNA molecules across the gel and a power supply generates charge difference across the gel. The DNA fragments can be visualised under UV light if ethidum bromide, a chemical that binds to DNA and fluoresces under UV light, is added during the process.
Uses: to find the size of the DNA fragment – detect base deletions/additions, to investigate mutations – sickle cell anaemia glu > val and to investigate DNA variation – DNA fingerprinting.

Front 4

Explain PCR and appreciate its fundamental importance in genetic testing

Back 4

Polymerase chain reaction (PCR) is used to diagnose many inherited diseases, detect the presence of tumours and the very early stages of infection by pathogenic microorganisms or viruses. It is able to amplify a very small and crude sample of DNA. It can also be used to discover novel sequences
The method of PCR is based on denaturation, hybridisation and DNA synthesis. The DNA sample is denatured by heat or Ph> 7 to separate the 2 strands, the DNA is cooled (annealing) and a pair of oligonucleotide primers are needed( forward and reverse) uniquely defining the region to be copied by one binding to a specific region on each of the template strands to initiate hybridisation. Thermostable DNA polymerase called Taq polymerase from thermus aquaticus is added to catalyse 5’>3’ DNA synthesis. These processes are repeated several times resulting in the exponential increase in amount of target DNA.

Front 5

Describe DNA hybridisation in southern blotting, microarray and FISH

Back 5

DNA hybridisation is also called southern blotting. DNA is digested by restriction enzymes to form DNA fragments. These fragments are separated according to size by gel electrophoresis. The separated DNA fragments are then transferred to a nylon paper by blotting. The nylon paper is placed in a sealed plastic bag along with labelled DNA probe in buffer. Hybridisation occurs where hydrogen bonds form between single stranded DNA fragments, the labelled DNA probe and specific complimentary sequences present on the nylon paper. DNA bands are visualised by autoradiography.
Southern blotting is used to investigate changes in gene structure eg deletion or duplication or to investigate gene expansion eg triplet codes – huntingtons disease and to investigate variation – DNA fingerprinting.
FISH – fluorescent in situ hybridisation is used to investigate DNA sequences on chromosomes inside the cell. Fluorescent probes for a specific gene/genes can be used or probes for specific DNA stretches on chromomes eg centromere, telomere. Chromosome painting is often used whereby each chromosome is visualised using a different coloured fluorescent probe.
Microarray – DNA passed over chip with lots of genes(probes) which hybridise with DNA/mRNA fragment , they label DNA for hybridization studies. Used to see concentration of gene.

Front 6

Explain what is similar in the studies of microarray, Southern blotting and FISH.

Back 6

All methods use the principle of DNA hybridisation. Southern clotting involves a fluorescently or radioactively labelled probe which hybridises to DNA fragments that have been size separated by gel electrophoresis. FISH technique use a fluorescently labelled probe to hybridise to DNA in chromosomes eg in situ = in the cell, in tissue. Microarray involves using 2 flurorescent probes to hybridise with DNA in 1000S of genes on a chip.

Front 7

Understand how PCR, restriction analysis and DNA hybridisation can be used in allele specific tests

Back 7

Allele specific probes/primers can be used in all techniques which are very sensitive to only one allele of a gene.
In PCR 2 primers are used which are very specific and only recognise one allele and not the other. Therefore PCR can test for the presence of one allele.
In southern blotting the probe, an allele specific oligonucleotide, is highly specific and will only recognise one allele.

Front 8

Describe the theory behind protein electrophoresis and how this technique can be used to provide information about protein structure

Back 8

Proteins are usually charged due to R group so if they are placed in an electric field they will go towards the relevant electrode: basic proteins will go the cathode and acid proteins will go to the anode. The migration of peptides is also based on their size and shape as smaller molecules can move faster through the gel. The proteins are applied to a porous support or gel which minimises diffusion.
2D –electrophoresis allows the separation of a complex mixture of proteins using both isoelectiric focusing and SDS-PAGE. Isoelectric focusing gel is placed on polyacrylamide gel and so proteins, already separated by isoeletric point move across the gel depending on their molecular weight, smallest can travel furthest.

Front 9

Which method would be used to separate proteins by solely molecular weight?

Back 9

Proteins can be separated purely by molecular weight by SDS-PAGE. The detergent sodium dodecyl sulphate denatures proteins and one SDS binds to 2 amino acids. The bound SDS has a large negative charge which masks the intrinsic charge on amino acids. Electrophoresis using a poluacrylamide gel takes place to separate the amino acids on basis of molecular weight and are visualised by adding a dye eg coomassie blue, which binds to proteins not the gel

Front 10

Which method would be used to separate proteins on the basis of isoelectric points?

Back 10

Isoelectric focusing is used to separate proteins purely on their isoelectric points and not molecular mass. A stable ph gradient, with decreasing Ph 9-3, is established in the gel after the application of an electric field. Then protein solution is added to gel and electric field is reapplied. Proteins separate on the basis of charge and migrate until they reach a pH equal to their pI. There is no net charge at pI and so stop migrating.

Front 11

Understand the basis for the use of enzyme assays

Back 11

In an enzyme assay the production of product or disappearance of substrate is measured and the activity gives an indication of whether that particular enzyme is present at normal levels. Enzyme assays are performed at optimal ph, temperature and ionic strength along with appropriate ions or co factors needed for enzyme action. High concentrations of substrate are used to ensure maximum activity by enzyme.
Enzyme assays are a diagnostic tool for metabolic diseases where an enzyme may be absent or present at low levels. The concentration of enzyme in serum is often measured as it is an indicator of tissue damage.

Front 12

Explain how antibodies can be used in immunoassays and western blotting to detect the presence of proteins.

Back 12

Polyclonal antibodies are produced by many beta lymphocytes, have many different types, only bind to one antigen(protein) and have many epitopes(amino acid sequences on a protein).
Monoclonal antibodies are produced by one type of beta lymphocyte, I identical antibody, specific to 1 antigen and 1 epitope.
Western blotting - proteins are separated by molecular weight by SDS-PAGE and then are transferred to nylon paper (membrane). The nylon paper is placed in a sealed plastic bag with specific antibodies. Primary antibodies bind to specific proteins on the paper and secondary antibodies, which bind to primary antibodies and are enzyme linked. The antibodies produce an immunoclot which is visible.
ELISA (enzyme linked immunoabsorbent assays) - used to find concentration of protein in complex mixture eg serum.
1. Immobilise first antibody on solid support
2. Apply protein containing solution eg serum
3. The antibody binds to the protein of interest and the other proteins wash away.
4. The secondary antibody, which is specific to the protein, is added and binds to the protein/ antibody complex
5. The secondary antibody is covalently linked to an enzyme so assaying for the activity of the enzyme by adding substrate and measuring the time taken for colour to change can measure the concentration of protein eg cortisol, insulin.
The secondary antibody is needed as although the primary antibody may be labelled, if the protein is in very low concentration the label may not be seen.

Front 13

Describe how enzymes are used as diagnostic tools.

Back 13

Enzyme assays are used to determine metabolic disorders in tissues by activity of enzyme and diagnosis of disease by concentration of serum enzyme.

Front 14

Name some important plasma hormones

Back 14

Cortisol: increased in crushing’s syndrome and decreased in addison’s syndrome
Noradrenaline and adrenaline: increased when phaeochormocytoma
Insulin: increased in obesity and decreased in diabetes ELIZA
TSH: increase in hypothyroidism and decreased in hyperthyroidism ELIZA
T4 &T3: increased in hyperthyroidism and decreased in hypothyroidism ELIZA

Front 15

Name some enzymes that if found in high concentration in serum, it is a marker for disease or tissue dammage.

Back 15

aspartate transaminase and alanine transaminase ( transamination of amino acids)- Liver damage
Creatine kinase, troponin, lactate dehydrogenase - MI / Ischaemic heart disease
Alkakine phosphorylase – secreted from osteoblasts in bone indicates high bone turn over , also released from bile duct, liver – bile stones.
Gamma glutamyl transferase – marker for liver damage increased by alcohol.
Pancreatic amylase, lipase – pancreatitis
Plasma cholinesterase – decreased in liver disease, inhibited in organophosphate poisoning
Acid phosphatase – marker for prostate cancer

Front 16

List the uses of all methods of molecular diagnosis:

Back 16

PCR – amplification of DNA fragment. Requires you have to have prior knowledge of the DNA sequence to use 2 forward and reverse primers . used check for absence or presence of a product and followed by gel electrophoresis or DNA sequencing or southern blotting (maybe with an allele specific oligonucleotide)
DNA gel electrophoresis – uses agarose gel, check for size of product, eg triple base deletion or addition. First the DNA is usually amplified by PCR but not too much as won’t be able to see the change in size. ( change from 70 to 73 can see, 570 to 573 can’t)
Southern blotting/hybridisation –uses nitrocellulose membrane, used to distinguish exact DNA sequence of interest from all the DNA fragments in a gel electrophoresis ( all very close, may be difficult to distinguish size of DNA fragment of interest). Might also be used with restriction enzymes when there is duplication of exons.
Restriction analysis – deletion or additions of exons could be detected by restriction enzymes ( would work if present, wouldn’t if exon not present – removal of restriction site) followed by southern blotting.
DNA sequencing – can be followed by PCR to determine the base sequence and thus see if a single base mutation etc appeared.
FISH – fluorescent in situ hybridisation – used to label a gene on a chromosome. eg 2 fluorescent probes may be used, one may be specific for the chromosome and one may be specific for a gene, if a chromosome has 2 labels, it has gene.
Chromosome painting – used to detect loss of material in chromosome. Different Fluorescent probes identify different chromosomes are different parts within a chromosome. Used along with comparative genome hybridisation (CGH) arrays.
Western blotting – uses nylon membrane. if mutation is in stop codon or splice site- lengthening or shortening of protein, electrophoresis of protein followed by western blotting, different size (immune clot formed – using primary and secondary antibodies)
Enzyme assay- analyses activity of enzyme
2D gel electrophoresis.- if change or loss in amino acid, 2d electrophoresis could be used to see if proteins is changed in charge or size. If a mutation in a stop codon/splice site- change in protein.

Front 17

Describe the different bands seen in a gel electrophoresis of haemoglobin in someone with sickle cell disease, sickle cell trait and normal.

Back 17

In sickle cell disease, there is a base substitution which causes the amino acid change glu to val in one of the beta polypeptides. Valine is a hydrophobic amino acid and so the protein is less negatively charged resulting in HbS moving to the positive electrode much slower. 2 bands of equal thickness of HbA and HbS would be seen in someone with sickle cell traits.