Lecture 1 Flashcards
Learning objectives
Function of different types of nucleases
Nucleases hydrolyse an ester bond within a phosphodiester bond between adjacent nucleotides.
Endonuclease: cuts in the middle of the DNA strand
Exonuclease: must begin at the end of a strand and progress into the fragment.
Restriction enzymes
Highly specific endonucleases. Recognises a specific motif - ONLY in the 5’->3’ direction.
Type II restriction enzymes are the most common, restriction and cleavage sites are the same.
Cloning of a DNA fragment into a plasmid
Restriction enzymes cuts the insert and the plasmid - so that they obtain identical ends - using two diffferent REs to prevent self-ligation. Ligases are used to ligate the insert into the plasmid.
Gel electrophoresis - both DNA and proteins
DNA: separates based on size due to the negative charge of DNA - migrates towards the positive pole.
Proteins: uses the detergent SDS to make the proteins migrate depending on size rather than shape.
Blotting
Detection of a specific sequence/gene using hybridized labeled probes that bind to the region of interest.
Southern: DNA - gel electrophoresis
Northern: RNA - gel electrophoresis
Western: proteins - SDS gel electrophoresis - proteins of interest are detected using antobodies.
Dideoxy (Sanger) sequencing
Uses ddNTPs which can be incorporated into a growing DNA strand because of their 5’ triphosphate group, but unlike the dNTPs, they do not include the 3’ hydroxyl group required to make a phosphodiester bond with the next nucleotide - thus they’ll terminate the DNA chain. Over time, all possible lengths of the sequence will be produced and these can be size separated by gel electrophoresis and the DNA sequence can be read from the shortest to the longest of the chains.
RT-PCR
Pairing PCR with a reverse transcription step (using reverse-transcriptase to reverse transcribe the RNA to cDNA) and then doing a regular PCR.
qPCR
Detects the products of PCR amplification during their synthesis and is more sensitive and quantitative than conventional PCR. For each cycle, the product formed is measured.
Reporter genes
Efficient to measure gene expression - you fuse the protein of interest with a reporter gene and measure the expression of the reporter.
Transcriptional fusions
The reporter gene is placed after the promoter of the gene of interest and before the SD sequence - thus the gene and the reporter each has their own SD sequence. Measures gene regulation at transcriptional level -> only measures the promoter strength - measures how much mRNA is produced.
Translational fusions
The reporter gene is placed after the promoter and SD sequence - measures the complete regulation of the gene - both promoter and SD strenght is measured - measures the amount of protein produced.
DNA microarrays
Test for up- or downregulated genes. We extract mRNA from two different cells (test and control) and do reverse transcription to convert it to cDNA which we label with two different colors. In the microchip, we have DNA strands that are complementary to the gene of interest. When the cDNA is introduced to the chip, we can detect gene regulation from the colors.
