Ch.20: Molecular Technologies Flashcards
What is gene cloning?
the technique of isolating and making many copies of a gene using a plasmid
What do restriction enzymes do?
protect bacterial cells from invasion by foreign DNA, particularly that of a bacteriophage and bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand
What is generated by the restriction enzymes?
sugar-phosphate backbone of DNA fragments with sticky or blunt ends (can be covalently linked together with DNA ligase)
What does reverse transcriptase do?
replicates RNA to DNA
What is the polymerase chain reaction (PCR)?
an in vitro method to amplify a specific sequence using directed DNA primers, DNA polymerase and dNTPS
What 3 steps does 1 PCR cycle include?
1.Denaturation (melting the ds target DNA)
2.Primer annealing (annealing of the PCR primers to the melted ss DNA)
3.Primer extension (amplification of new DNA by DNA polymerase)
When is the 1st complete proudct formed?
After the 3rd cycle of PCR
What is Reverse transcriptase PCR (RT-PCR) is used for?
to convert RNA to dsDNA
What is Real-time PCR or quantitative PCR (qPCR) used for?
to quantify the amount of mRNA that is expressed from a specific gene in a sample
What does the dideoxy sequencing method involve?
involves the replication of DNA by a DNA primer, DNA polymerase and deoxyribinucleotides (dNTPs) in
combination with dideoxyribinucleotides (ddNTPs) that terminate replication
What is the 2nd method used for DNA sequencing?
base-specific cleavage of DNA
by chemicals
How do you read the traditional Sanger DNA sequencing gel?
Resulting DNA sequence read from the smallest fragment to the largest fragment, since this corresponds to the order of the sequence from the primer
Describe the CRISPR-Cas gene editing system
A single RNA in which the tracrRNA and crRNA are linked together called single guide RNA (sgRNA) can be created and used to do gene editing in any cell type. A spacer region is designed that has homology to the targeted DNA to be edited
What are Southerns?
The first blotting method to detect regions of DNA homology
What are Northerns?
used to identify a specific mRNA and has many uses:
1. It can determine if a specific gene is transcribed in a particular cell type
2.It can determine if a specific gene is transcribed at a particular stage of development
3.It can reveal if a pre-mRNA is alternatively spliced
