class-1132

• Situ sequencing


  Can be performed on fixed cells
• Types of nucleic acid polymorphisms


  • Short tandem repeats: Copies of 3,4,5 nucleotides repeat
  • Single nucleotide polymorphisms: point mutation that's carried by some individuals from a population
  • Restriction fragmented lengths: Mutations of alterations on restriction sites where changes fragments when the DNA Is cut
• General methods used to prepare a DNA Library for NGS

  1. Fragmented DNA

  2. End repair: which has its corrosive ends that's repaired from an enzyme

  3. A-tailing: The enzymes will perform the A-tailing

  4. Adapter and adapter extension: which gives an overhang

  5. PCR: fragments are amplified generating many copies
• Ion semiconductor


  NGS method that does not use a light/camera to detect
• Dideoxy sequencing


  Chain termination sequencing/sanger sequencing
• Chip


  Mapping the modifications of the histones. Used to investigate the specific protein DNA interactions
• Why use 2D instead of the standard one

  In 2D its study's the present and absence of proteins. It uses a pH gradient where the proteins migrate towards the one side to another. The isoelectric strip is used where proteins move towards it. This is a better way to see the mixture of proteins
• BLAST suite


  • BLASTN
  • BLASTP
  • BLASTX
• N and X


  N is nucleotide and p are protein
• Multi omics


  Used in disease research to identify biomarkers. Applications Genomics, transcriptomics, proteomics, metabolomics, epigenomics
• Comparative


  Studies and identifies the gene sequence
• Proteomics


  Study of a set-in proteins that's expressed by a genome
• Transcriptomics


  Study of mRNA as a mechanism to monitor gene expression
• Metabolomic


  Study of metabolites in present of cell through life cycle
• Epigenomics


  Study of cause and impact of epigenetics change to a genome
• Northern blotting


  Using an agarose gel to detect the presence of RNA. A labeled fluorescent is added and then RNA can be detected
• 3rd gen nanopore sequencing method

  Uses nanopores that's embedded from an electric current. where dsDNA fragments mixed with an enzyme that's moved through. The nanopore. The information used is interpret the order of nitrogen bases
• How the use of restriction enzymes can allow for the determination of the methylation status at a specific target sequence of interest


  RE can cut DNA only if it's not covered in methyl. Methylation packs the DNA tightly and nothing can bind. This can help us see if certain areas of DNA are tagged with methyl or not. After DNA is cut, use PCR to make a lot of copies. Use qPCR to look closely at the copied DNA. Fluorescent probes are used. Two sets of qPCRs are performed= 1 targets the methylated and the other the unmethylated allele. Can see and compare the different regions of the DNA
• RT-PCR


  RT-PCR is named reverse transcriptase where it binds to single stranded RNA molecules to generate complementary DNA nucleotides. For the second part the CDNA has those coding portions that will synthesize into the mature mRNA
• General steps of Gibson assembly

  We first start with two overlap ends. Endonuclease such as ends gets chewed off from the 5' leaving with two partially digested strands. Polymerase extends the gaps leaving the ligase to fix the nicks
• Massively parallel sequencing


  It means it sequences the whole genome generating lots of fragments at once
• SMRT sequencing


  The library is bound to a zero-wavelength chip. The DNA pol and template is fixed to the bottom of the chip. The dNTPs are added at once with different color fluorophore. A camera below detects the color. The dNTP is cleaved with the PPi removing the fluorescent single in the detectable window
• Nanopore sequencing


  • It is unique because its not sequenced from synthesis and no DNA polymerase is involved. One advantage is it uses real time and sequences long sequences
• In situ single cell RNA sequencing

  Does not require conversion to RNA to cDNA
• Genotyping


  It is the differences of the DNA sequences between induvial at select regions of the genome. It can be used for crop trait improvement and seed quality control
• Massively parallel sequencing


  It sequences the entire genome
• Role of antibodies locating acetylated histones using Chip method

  The antibodies are bind to the histone modification where its precipice chromatin fragments used to analyze the locations
• Difference between regular agarose electrophoresis and pulsed field gel electrophoresis

  In the pulse it can analyze large fragments and its useful when doing genotyping and fingerprinting
• Process of microarray and how to interrupt the results

  The mRNA is copied in RT PCR where a fluorescent tag is used for the results of two types of cells. The green is healthy and red is diseased. The cDNA is washed off and is detected the color patterns by a UV light. Black is no expression while yellow is the same expression
• Types of NGS


  • illumina
  • pyrosequencing
  • ion semiconductor
• Mass spectroscopy


  Molecules are ionized through a magnet. The protein is broken into small segments where the whole protein can be obtained this is good for study of mutations
• Xray


  The protein is a ribbon structure. It is first started with a crystal this crystal is passed through an Xray that's when the computer comes along
• Types of protein microarrays

  • Analytical: Identify diverse proteins preset within the cell by fixing the microarrays
  • Functional: studies binding proteins by fixing purified proteins in a native structure
  • Reverse phase: proteins from the cell lysate fixed to a microarray
• How BLAST works


  It looks for two adjacent word pairs. It extends the similarity in either direction of the sequence. The high score subject pairs are ranked
• Homology searching


  Searching for one genome at a time. Sequence
• Ways bioinformatic tools can be used to identify genes in a genome

  • ORF scans which is a series of nucleotides that start with a start codon and end in a termination codon
  • Codon bias will help to locate where the genes are in a genome
  • Exon intron boundaries
• Types of primers


  • Standard PCR: amplification of two primers
  • Real time PCR: quantity of product
  • RT PCR: amplifies and isolates
  • Assembly PCR
  • Colony PCR: screening bacterial colonies
• Using the bisulfite treatment method to determine the methylation status of a target DNA fragment


  If it us unmethylated the C will end up turning into a T and if it is methylated the C will stay as a C
• Primers used when doing RT-PCR to create a cDNA library


  The poly T nucleotide will hybridize to the poly A tail. The RT will extend to build a cDNA. Once the cDNA is made a TaqMan uses the second set of primers. which amplifies the cDNA
• qPCR SNP genotyping


  They are used to design TaqMan probes each SNP location is linked with a different color