L7,8,9 - Molecular Biology Techniques Flashcards
What does plasmid contain?
Origin of replication, antibiotic resistance genes and multiple cloning sequence (MCS)
What does RFLPs stand for?
Restriction fragment length polymorphisms - changes in DNA sequence at a restriction site changes the sizes of restriction fragments
Expression vectors
are plasmid cloning vectors designed to allow expression of cloned genes in bacteria with the purpose of producing large quantities of encoded protein
Eukaryotic expression vectors
are plasmid cloning vectors designed to allow expression of cloned genes in eukaryotic tissue culture cells
In dideoxynucleotide sequencing, what gel is used?
Polyacrylamide gel
What is PCR?
It is a powerful technique that has been recently developed to allow selective amplification of specific regions of DNA - make many copies of specific DNA fragments without need to use cloning vectors or restriction enzymes
How many steps are involved in PCR and what are they?
3 steps - Denaturation, Primer annealing, Extension (DPAE)
Denaturation
temperature: 95 degrees to denature the DNA into single stranded (30 seconds)
Primer Annealing
temperature: 58 degrees to allow primers to hybridise to their complementary sequences in the target DNA (30 seconds)
Primer extension
temperature: 72 degrees to allow Taq polymerase to synthesise DNA (1 minute)
What are the limitations of PCR?
1) some informations about the nucleotide sequence pf that target DNA must be known in order to synthesise primers
2) Minor contaminations of sample DNA from other sources can cause problems
3) PCR cannot amplify long segments of DNA, only those that are relatively short (5-10kb max)
Reverse transcription PCR (RT-PCR)
is used to study dene expression by examining mRNA production by cells or tissues
Quantitative real-time PCR (qPCR)
allows researcher to quantify amplification reactions as they occur in real time to identify amount of DNA in a sample
Nucleic acid hybridisation
it is a method to allow identification of nucleic acids fragments or clones containing specific sequences
What is used as probe in nucleic acid hybridisation?
ssDNA
DNA probe is labelled by using DNA polymerase to incorporate labelled dNTPs. dNTPs can be labelled using…
1) radioactively using 32P
2) attaching a fluorescent molecule
3) attaching digoxygenin
What can Colony Blot hybridisation use for?
- it can be use to identify clones in a cDNA library containing specific DNA sequences
- Grow colonies of bacteria, each derived from single bacteria which contain an individual plasmid clone from cDNA library
Types of nuclei acid hybridisation
Colony Blot hybridisation, Southern Hybridisation, Northern Hybridisation, In Situ hybridisation and DNA microarray
Southern Hybridisation Blot
Allows identification of DNA molecules containing specific sequences
Northern Hybridisation Blot
- Allow identification of RNA molecules containing specific sequences
- Most often used to identify if a specific gene is expressed, i.e. transcribed into mRNA, in a particular tissue or sample
in situ hybridisation
- hybridising a probe directly to RNA without blotting
- Probe is hybridised to the mRNA transcript in situ i.e. in the place where the transcript is made
DNA microarrays
- They are modern devices which use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample
- uses a reverse approach whereby ALL the mRNA from a sample is converted to cDNA, fluorescently labelled and used as probe
- this probe is applied to the array which contains spots where single stranded DNA from each gene in the genome is laid out in an array
- enables the ability to simultaneously identify all the genes expressed in a particular sample
