L24- Molecular techniques Flashcards
why do we use restriction analysis?
- To investigate the size of DNA fragments e.g. small deletions
- To investigate mutations e.g. Sickle cell disease
- To investigate DNA variation e.g. DNA fingerprinting
what enzymes are used for restriction analysis
endonucleases
what produce endonucleases
bacteria
specific restriction endonucleases
recognise and cut specific DNA sequences (phosphodiester bonds) (at restriction sites)
restrictriction sites are mostly
palindromes
when restriction endonuclease cut at palindromic restriction sites we will ge an
uneven stagger
- producing sticky ends
- 5’ overhang- can be joined back together using ligases
restriction endonuclease are a type of
molecular scissors
Repertoire of restrictions endonucleases at our deplorable
e.g. BamHI, EcoRI, Pstl
how can restriction sites be predicted
using software
what alter restriction sites, meaning endonuclease will be useable to cleave DNSA
mutations
gene cloning uses
plasmids e.g. from E.coli
outline gene cloning
- Isolate relevant gene of interest following digestion with restriction enzymes
- Insert gene of interest into plasmid vector (recombinant DNA molecule)
- Introduce recombinant DNA molecule into suitable host cell e.g. E.coli
- Identify and isolate the clone containing the DNA of interest
plasmids
- Small circular dsDNA
- Found in bacteria
- Mini chromosomes
- Can transfer to other bacteria
- Often carry antibiotic resistance genes
why clone human genes
- Make useful proteins e.g. insulin (put into expression vector and gives us human insulin protein)
- To find out what genes do e.g. HTT
- Genetic screening e.g. Huntington’s, BRCA1/2, CF
- Gene therapy e.g. CF
polymerase chain reaction is a method of
amplifying small amounts of target sequences rapidly
requirements for PCR
- Thermostable DNA polymerase (Taq)
- Pair of primers (forward and reverse), uniquely defining the region to be copied
- Thermocycler
why use PCR?
- Amplify specific DNA fragment
- To investigate single base mutations e.g. Tay sachs and sickle cell
- To investigate small deletions or insertions
- To investigate variation, genetic relationships e.g. DNA profiling
process of PCR simple
- Denaturation
- Annealing stage
- Extending stage
denaturation
(95 degrees): DNA denatures (breaks H bonds and becomes ss)
annealing
(50-56 degrees) - specific primers added bind to complementary sequence
extending
(72 degrees): Taq polymerase added and DNA synthesis
PCR and copying of DNA is
exponential increase in DNA
primers are included in
the end product
DNA electrophoresis
DNA is negatively charged and will move towards the anode if placed in an electric field- DNA fragments can be separated on the basis of size (or shape).
what happens to DNA before electorphoresis
- cut by restriction endonuclease- only specific genes being look at
DNA electrophoresis separates DNA on the basis of
size (or shape)
requirements of gel electrophoresis
- Gel- a matrix that allows separation of DNA fragments
- Buffer- allows charge on the DNA samples across the gel
- Power supply- generates charge difference across the gel
- Stain- to identify the presence of the
protein electrophoresis
- Proteins can be separated based on size, shape or charge
why can proteins be separated using electrophoresis
Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric filed.
e.g. serum proteins
if we want to separate the proteins just on the basis of size
we add detergent SDS (removes charge)
adding detergent to proteins
o Makes a folded protein in a linear polypeptide chain
requirements of gel electrophoresis
- Gel- a matrix that allows separation of the protein sample
- Buffer- maintains charge on the protein samples
- Power supply- generates charge difference across the gel
- Stain – to identify the presence of separated proteins
when a protein electrophoresis is finished, what can be used to identify unknown proteins
Mr standards- to compare size
protein identification
proteomics
proteomics example
- Digest proteins with trypsin
- Perform mass spec
- Generate list of peptide sizes
- Use database of predicted peptide sizes for known proteins to identifying the specific protein
immunoassays
use of antibodies in the identification of protein
antibodies have unique structures which recognise specific antigens
paratopes which recognise epitomes on an antigen
types of antibodies
polyclonal
monoclonal
polycolonal
- Produced by many b lymphocytes
- Lots of diff antibodies which recognise diff epitopes on the same antigen
- Most commonly used in diagnosis
monoclonal
• Recognise a single epitope on a single antigen
how can we use antibodies (name 4 techniques)
- Western blotting
- Southern blotting
- Northern blotting
Weston blotting allows
identification of particular proteins from mixture of diff proteins
ELISA can be
- Indirect
- Direct
- Sandwich
ELISA used to
- Measure the conc of proteins in solution:
o Insulin
o Cortisol
o TSH
‣ The more antibody that binds the more protein present
outline how indirect ELISA works
1) Antigen coated to well
2) specific antibody binds to antigen
3) enzyme- linked antibody binds to specific antibody
4) Substrate is added and converted by enzyme into coloured produced, the rate of colour formation is proportional to the amount of specific antibody
enzyme assay
way of calculating rate of an enzyme catalysed reaction
how do enzyme assays work
Can measure rate for activity of an enzyme- by adding substrate and measuring conversion of substrate to product over time = rate
continuous assay
- Spectrophotometer
• Chemoluminescence
discontinuous assay
- Radioactivity
* Chromatography
measurement of enzymes can be used to diagnose
metabolic disorders and in diagnosing disease
clinically important serum enzymes for liver disease
aspartate transaminase
alanine transaminase
clinically important serum enzymes for pancreatisis
amylase and lipase
clinically important serum enzymes for liger damage (increased by alcohol)
Y-glutamyl transferase
clinically important serum enzymes for bone disroders
alkaline phosphatease
clinically important serum enzymes for bone disorders
alkaline phosphatase
CK-1
brain
CK-2
BM (myocardium)
CK-2
MM- skeletal
gold standard for diagnosis of an MI is the measurement of
cardiac troponin I (cTnl) by ELISA
