Gene expression analysis

• What techniques are used for mRNA expression analysis

  Northern blotting.

  RT-PCR.

  Microarray
• What techniques are used for protein expression analysis

  Western
• What types of probes are used for nucleid acid analysis

  Radioactive, chemical or fluorescent
• Describe how a radioactive probe is detected

  Using autoradiography
• Describe how a chemical probe works

  An antigen is incorporated into the DNA. It is labelled with an enzyme-linked antibody which produces a coloured or chemiluminescent compound
• What techniques make up RT-PCR

  Reverse transcriptase and PCR to make cDNA

  Gel electrophoresis
• What does RT-PCR compare

  Gene expression in different samples
• Describe how RT-PCR works

  Reverse transcriptase converts mRNA to single-stranded DNA using a poly-dT primer.

  RNAse H degrades mRNA.

  DNA polymerase uses a primer and DNTPs to synthesize the second DNA strand.

  The cDNA is amplified using PCR and analysed using gel electrophoresis.
• What does RT-PCR produce

  cDNA which has no introns
• what is the other name for a microarray used for gene expression

  RNA-seq
• Describe how RNA-seq works [Microarray]

  mRNA is converted to cDNA using RT-PCR. They are then labelled using a fluorescent dye and hybridized to the microarray. The microarray is then washed to remove unbound material and analysed using technology.
• What does black indicate in a microarray

  Neither sample expressed the genes
• How is cluster analysis used

  It identifies genes with the same expression pattern.

  Microarray is conducted on cell samples exposed to different conditions.
• What does in situe hybridisation do

  Determines where and when a single gene is expressed
• What is epigentics

  These are heritable changes in gene activity which are not caused by changes in the DNA sequence.
• How is DNA packed

  It is wrapped around histones to form chromatin.
• What is methylation of the genome

  It is the addition of a methyl group [CH3] to the DNA. This inhibits transcription by forming heterochromatin
• What form of DNA is tightly wound

  Heterochromatin
• What is phosphorylation of the genome

  The addition of a phosphate group to the histones
• What is acetylation of the genome

  The addition of an acetyl group [- COCH3] to the histones. This allows for transcription as it forms euchromatin.
• What form of DNA is wound loosely

  Euchromatin
• Describe 3rd generation sequencing

  They allow for longer reads of a single molecule to be sequenced in real-time.

  They also allow you to detect DNA modification.
• What does gene expression tell you

  The amount, time or location that mRNA and proteins are produced
• What are the two forms of 3rd generation sequencing

  PACBio and Oxford Nanopore
• Why are 3rd generation sequencing techniques able to detect DNA modifications

  They do not use PCR to amplify the DNA. This is important because when DNA is amplified with PCR the modifications are lost.
• Describe how PACBio sequencing works

  The DNA is fragmented and ligated to adapters. It is then mixed with DNA polymerase and added to a cell consisting of multiple chambers.

  Fluorescently labelled Deoxyribonucleotide triphosphates are added.
• What is the result seen in PACBio sequencing


  When the DNA polymerase incorporates the fluorescently labelled deoxyribonucleotide triphosphates, fluorescent light is produced and detected using a camera. This records the wavelength and position of the incorporated bases.
• Describe how Oxford Nanopore sequencing works

  The flow cell consists of protein nanopores, which are embedded in a synthetic membrane. The DNA molecules are attached to adapters.

  Motor proteins unwind the DNA, and an electric current moves the negatively charged DNA towards the nanopores.
• Describe the result of Oxford nanopore sequencing

  As DNA moves through the nanopores it causes disruptions to the current.

  Each nucleotide will produces a characteristic signal which can be recorded.