Introduction to Molecular Techniques Flashcards
What techniques are involved in analysis of DNA at the gene level?
Restriction enzymes, DNA gel electrophoresis and PCR
Also southern hybridisation, microarray, PCR variations
What techniques are involved in analysis of protein?
Protein electrophoresis, immunoassays and enzyme assays
What technique is involved in analysis of DNA at the nucleotide level?
DNA sequencing
What techniques are involved in analysis of DNA at the chromosome level?
Karyotyping and FISH
What are restriction enzymes?
Enzymes that cut a DNA molecule at a particular place
What bonds do restriction enzymes cut?
Phosphodiester
What do restriction enzymes recognise and cut?
Specific DNA sequences (restriction sites) - mostly palindromic
What restriction site does EcoRI recognise?
GAATTC
What does EcoRI digestion produce?
Sticky ends (complementary base pairs)
How does DNA gel electrophoresis separate DNA fragments?
Based on their size - DNA is negatively charged due to the phosphate groups so they will move towards the positive electrode. Smaller fragments move further.
What are the requirements for gel electrophoresis?
- Gel - matrix allowing separation of fragments
- Buffer - allows charge on the DNA samples across gel
- Power supply - generate charge difference
- Stain/detection - identify presence of separated DNA (fluorescent markers)
Why would we use restriction analysis?
- To investigate size of DNA fragments to see if there are any changes in DNA (eg small deletions)
- To investigate mutations (eg sickle cell disease)
- To investigate DNA variation (DNA fingerprinting)
- To clone DNA
What are the four basic steps of gene cloning?
- Isolate relevant gene following digestion with restriction enzymes
- Insert gene of interest into plasmid vector (recombinant DNA molecule)
- Introduce recombinant DNA molecule into suitable host cells (eg Ecoli)
- Identidy and isolate the clone containing the DNA of interest
Why would we clone human genes?
To make useful proteins eg insulin
To find out what genes do eg HTT
Genetic screening eg Huntingtons
Gene therapy
What does reverse transcriptase do?
Converts RNA to cDNA
What is the enzyme used in the Polymerase Chain Reaction?
Taq polymerase
What does PCR require?
Taq polymerase
Primers
Nucleotides
Why is taq polymerase used in PCR/
It is thermostable
What is the purpose of PCR?
To amplify DNA
What are the basic steps of PCR?
- At temp of 90 degrees, DNA denatures and hydrogen bonds are broken
- At temp of 60 degrees, primers anneal
- At temp of 72 degrees, TAQ polymerase adds nucleotides to complementary strands
In PCR, what defines the region to be copied?
Pair of primers - forward and reverse
Why would we use PCR?
To amplify a specific DNA fragment
Investigate single base mutations
Investigate small deletions or insertions
Investigate variation and genetic relationships
How can proteins be separated in gel electrophoresis?
Basis of size, shape or charge
What technique is used for the separation of proteins on the basis of size?
SDS-PAGE (use of detergent)
What technique is used for the separation of proteins on the basis of charge?
Isoelecric focusing (IEF)
Proteins migrate until they reach a pH equal to their pI (no net charge at pI so stop migrating)
What technique allows the separation of a complex mixture of proteins?
2D-PAGE (important for diagnosing disease states in different tissues)
Firstly separate on basis of pH, then as there may be multiple proteins on each band, separate on basis of size
How are proteins identified?
- Digest protein with trypsin
- Perform mass spectrometry
- Generate list of peptide sizes
- Use database of predicted peptide sizes for known proteins to identify the protein
What amino acids are cleaved by trypsin?
Lysine, Argenine
What amino acids are cleaved by staphylococcal protease?
Aspartate, glutamate
What amino acids are cleaved by chymotrypsin?
Tyr, Trp, Phe, Leu
What amino acids are cleaved by endopeptidase Lys-C?
Lysine
What does hydroxylamine cleave?
Asp-Gly bonds
What does cyanogen bromide cleave?
Methionine
What does western blotting detect?
Specific proteins
What technique can be used to measure the concentration of proteins in solution eg hormones?
Enzyme-linked immunoabsorbent assay (ELISA)
What assay method is used in the measurement of the concentration of cortisol?
RIA (radioimmunoassay)
What assay method is used in the measurement of the concentration of insulin?
RIA, ELISA
What assay method is used in the measurement of the concentration of TSH?
RIA, ELISA
What assay method is used in the measurement of the concentration of T3 and T4?
RIA, ELISA
What condition is cortisol increased in?
Cushing disease
What condition is cortisol decreased in?
Addison disease
What condition is insulin increased in?
Obesity
What condition is insulin decreased in?
Type I diabetes
What condition is TSH increased in?
Hypothyroidism
What condition is T3/T4 increased in?
Hyperthyroidism
Why does SDS-PAGE separate on basis of size?
SDS disrupts tertiary structure of proteins to bring the folded proteins down to linear molecules. It also coats the proteins with uniform negative charge
Why does SDS-PAGE separate on basis of size?
SDS disrupts tertiary structure of proteins to bring the folded proteins down to linear molecules. It also coats the proteins with uniform negative charge
How can glucose concentration be measured?
Glucose oxidase
Test strips - H2O2 converted to coloured dye
What enzymes are markers for liver damage/disease?
Aspartate transaminase and Alanine transasminase (AST and ALT)
gamma-glutamyl transferase - increased by alcohol
What enzymes are markers for pancreatitis?
Amylase/lipase
What enzyme is a marker for bone disorders?
Alkaline phosphatase
