Molecular Techniques Flashcards
What produce endonucleases
Bacteria
What do restriction enzymes do
Recognise and degrade foreign DNA
Which electrode do DNA fragments travel toward in electrophoresis and why
Positive as DNA is negatively charged
What sized fragments travel the furthest
Small
What does DNA gel electrophoresis do
Separate fragment based on size
How are genes cloned
Isolate specific gene with restriction enzymes and insert into plasmid vector to place into bacteria to replicate naturally
What are plasmids
Small circular DNA
Give an example of when human genes are cloned
To make human insulin
What enzyme concerts mRNA into cDNA
Reverse transcriptase
What disease is currently being treated using gene therapy
Cystic fibrosis
What enzyme is used in PCR and why
Taq Polymerase as it can work at higher temperatures
What is the first step in PCR
Heat strands to 95 degrees to denature DNA to give single strands
What is the second step of PCR
Cool DNA to 55 degrees to allow DNA primers to bind
What is the final step of PCR
Heat to 72 degrees to allows taq Polymerase to bind and add nucleotides from the 3’ end.
Why is PCR used
Look for mutations, to see if there’s a loss or gain of restriction fragments or to see size of product
Why can you use PCR to determine if someone has sickle cell anaemia
In sickle cell the mutation changes a restriction site and so this site will not be cut like it would in normal DNA
What does PCR result in
Lots of copies of a specific piece of DNA
What is protein gel electrophoresis
Separation of proteins
What different types of protein electrophoresis are there?
SDS page, isoelectric focussing and 2D page
What is SDS page
When proteins are denatured, given a negative charge and separate due to size as they migrate to the positive electrode
What is 2D page separation
Separation of proteins based on size and charge
What is isoelectric focussing
Separation of proteins based on charge. Protein migrate until they reach a pH equal to their pI
What is proteomics
Protein identification
How do you carry out proteomics
Digest protein, perform mass spectrometers and identify protein based on peptide sizes
What Techniques uses antibodies instead of DNA hybridisation
Western blotting
What is ELISA
A technique to detect protein concentration in a mixture by using antibodies to bind to the protein
What is an enzyme assay
A technique used to measure enzyme activity by measuring appearance of product or disappearance of substrate
The concentration of which enzyme increases during a heart attack
Creatine Kinase
What are monoclonal antibodies
Antibodies that can bind to 1 epitope/protein only
What are polyclonal antibodies
Antibodies that can bind to multiple epitope in the same antigen
Outline DNA hybridisation
When a double stranded DNA is heated and denatured and then labelled, complementary markers are allowed to anneal to the single strands.
Outline southern blotting
Separate DNA using electrophoresis then transfer to membrane, denature and hybridise with fluorescent
probes to visualise DNA fragments
What is northern blotting
Uses DNA in a method similar to northern blotting but detects RNA so tells you gene expression
What is western blotting
Using antibodies to visualise proteins
What produces DNA probes
Oligonucleotide synthesiser
What are ddNTPs
DNA nucleotides that have 3 phosphate groups and no OH group
What happens when ddNTPs are incorporated into the growing chains
They stop the chain from growing as the lack of OH stops phosphodiester bonds being made
What technique uses ddNTPs
DNA sequencing
What can you do with micro arrays
Investigate many genes at one time
Outline micro arrays
You compare gene expression from a healthy and diseased patient by mixing samples with hybridisation probes and look at colour to determine expression
What regions are compared during DNA fingerprinting
Minisatellites
What are minisatellites
Non coding regions of DNA
Why are minisatellites compared for DNA profiling
Everyone has different amount of different minisatellites
What is karyotyping
Displaying metaphase chromosomes
What is FISH
Making probe which are specific for a whole chromosome or gene so that these areas can be visualised
What ethical issues are there with investigating the genome
Potential for terminating babies, can’t identify all mutations, can lead to future personal consequences (e.g with genetic diseases you may or may not have children )
What are reasons for testing someone’s genome
To diagnose conditions, for prenatal screening for pregnancy decision and to see if someone’s a carrier
What is a primer
Short section of DNA that acts as a starting point for DNA synthesis as Polymerase binds here
What is reverse transcriptase PCR
Starting with an mRNA strand, producing the cDNA strand and amplifying it
