Lecture Cycle 10- DNA technologies

• What is the process of PCR? What are the reagents? What temperatures are needed at each step?

  - step 1: denaturing, start with parent DNA and break the hydrogen bonds to create two separate strands, temp = 95

  - step 2: annealing, add forward and reverse DNA primers, DNA poly, dNTPs and Magnesium Chloride, temp = 55

  - step 3: synthesizing, making new DNA strands, temp = 72
• What is the purpose of PCR?

  take a segment of DNA and amplify the target
• How is RT-PCR used to quantify the abundance (amount) of mRNA, also known as gene expression levels or transcript levels?

  measure the amount of cDNA
• What is the process of reverse transcription?

  mRNA is reverse transcribed to create cDNA [single stranded] using reverse transcriptase
• How PCR is used for DNA profiling? (STR regions, sex determination,

  - amplify the 13 specific STRs used during DNA profiling, using an electropherogram to analyse the gel you can match up the peaks with the standard

  - sex determination: AMEL gene is longer on the Y
• How do you read an electropherogram?

  match the peaks from the sample to the standard, all 13 peaks must match
• What is the difference between mRNA and cDNA?

  cDNA is the single stranded DNA sequence that is complimentary to the mRNA
• What is the process of RT-PCR? What does the oligo(dT) primer do?

  - extract RNA and treat with DNase

  - oligo(dt) primer binds to poly A tail, this is how we "fish" for mRNA

  - reverse transcription has DNA polymerase activity at the primer and degrades RNA into cDNA

  - you then sythesize the cDNA and perform PCR
• What is RT-PCR used for? What can we then determine?

  measure the gene expression level
• How is it possible to express a human gene in a bacterial cell?

  - using RT

  - take cDNA of the human gene and insert it into a bacterial plasmid

  - insert this plasmid back into the bacteria

  - bacteria replicates, also replicating the human gene
• How can bacteria synthesize human insulin?

  - insert the cDNA for the human insulin gene into a bacterial plasmid

  - insert this plasmid into the bacteria

  - when the bacteria replicates, the human gene will also be replicated
• What are the mechanisms within the adaptive immune system of bacteria? What are the 5 steps that a surviving cell went through?

  - the two phases of adaptive immune systems; immunization and defense

  - step 1: viral DNA processing; cas processes the viral DNA

  - step 2: the processed viral DNA is then incooperated into the bacterial DNA

  - step 3: transcription occurs and spacers and tracrRNA are expressed

  - step 4: Pre- crRNA processing

  - step 5: CRISPR-Cas9 is guided to the viral DNA and cleaves it
• What are the two ways a cell repairs double stranded breaks?

  - NHEJ: non-homologous end joining

  - HDR: homology directed repair
• What is the key to remember about NHEJ?

  it is extremely error prone and sloppy
• What is HDR?


  - homology directed repair

  - repair using a custom made template
• What is a PAM sequence? What is the PAM sequence in Cas9? Where is it in relation to the cut site?


  - essential sequence needed for Cas to cut

  - NGG

  - downstream of the cut site
• What is an essential sequence needed within Cas9 in order for it to cut?

  PAM sequence
• How are both NHEJ and HDR used by CRISPR-Cas9 to disrupt, correct mutations or insert normal copies of genes?

  - NHEJ: is used by CRISPR-Cas 9 to disrupt the gene

  - HDR: is used by CRISPR-Cas 9 to correct a mutation or insert a normal copy of the target gene
• What is the difference between a dCas, nCas, or Cas?

  - dCas: dead, does not cut

  - nCas: nicking, nicks one strand only

  - Cas: double stranded cut
• How does base editing work? What is the pathway?

  - gene editing specific for one base pair

  - deamination of target base in ssRNA

  - nCas nick occurs in strand not undergoing chemistry

  - use UGI to inhibit to inhibit natural repair

  - mismatch mutation, leading to a base change after replication
• How is CRISPR being used to advance immunotherapy?

  - use CRISPR to knock out PD-1 of normal T cells

  - expansion of edited T cells

  - edited T cells can recognize cancer cells
• What is PD-1?


  - protects tissues from autoimmune attack

  - causes chronic infection and tumor progression