Describe microarray hybridization technique
Glass slides with probes that identify each gene, fluorescently labeled
DNA cloning uses cloning vectors and PCR. What two enzymes are required?
Restriction enzymes and DNA ligase
What are the features required for a vector in DNA cloning? (3)
origin of replication multiple cloning site (restriction sites) ABX resistance gene (selectable marker)
DNA cloning produces two types of DNA libraries. Describe differences.
gDNA - chromosomal DNA, digested then fragments can be cloned cDNA - DNA transcribed to RNA then reverse transcription then DNA cloned
Describe temperature steps for PCR
1) Denaturation (94C) 2) Annealing (depends on primer) 3) Extension (72C)
Real time PCR uses fluorescent probes/dyes to detect and quantify ____ in real time
DNA, if reverse transcriptase used, can quantify mRNA
Which of the following is not a use of PCR? a) genetic testing b) clone gDNA c) forensic/identity screens d) identify translocation
What is analyzed in Western blot and how?
proteins, travel based on molecular weight
Regulated gene expression uses a ______________ to turn genes on and off
miRNA and siRNA both cleaved to mature form by _______
miRNA is _______ expressed while siRNA is ________ expressed
Describe the mechanism of interference by siRNA
dsRNA processed by dicer --> siRNA comes with RISC --> RISC cleaves siRNA duplex --> RISC activated --> RISC guided to target DNA strand and cleaves
What are the four requirements for DNA sequencing?
free 3'OH group, DNA polymerase, primer, template
Describe Sanger sequencing
ddNTPs incorporated to initiation chain termination - no free 3' OH end
How would this gel (result of sequencing) be read?
From bottom up (ATGCT etc)
Describe an antisense molecule
Bind to specific complimentary sequences and inhibit gene expression
Automated sequencing accelerated the human genome project. What is the major limitation of this and how is it overcome?
LIMITATION = only short sequences can be obtained in a single rxn
Overcome by shotgun sequencing - gDNA broken into fragments then sequence rebuilt
Shotgun sequencing leads to creation of genome maps. Describe the difference between genetic and physical maps.
Genetic maps produced by genetic techniques
Physical maps produced by molecular bio techniques
- includes restriction mapping, FISH, and STS mapping (positions of unique sequences)
Genome mapping shows genes and DNA markers such as SNPs, ssLPs and RFLPS. Describe RFLPs.
Restriction Fragment Length Polymorphisms= result from polymorphic restriction sites. Important for genetic testing to indicate disease locus, can analyze via Southern Blot or PCR
Describe homologous recombination in producing transgenic animals
Plasmid vector construct identical except for TK and target gene which produces target locus after homologous recombination. Southern blot will show proteins.
Describe Cre/IoXP use in site specific recombination and conditional gene targeting
Targeted gene floxed in between two recombinase binding sites such as IoxP, Cre is recombinase that can eliminate gene under certain conditions (tissue, time)
Pronuclei injection can also be used to generate transgenic animals. Describe.
Desired gene injected into fertilized egg --> egg implanted
Describe the two components of CRISPR gene editing
Cas9 protein - molecular scissors
guide RNA - guides cas9
Double strand breaks caused by Cas9 can be repaired in two mechanisms. Describe.
Non-homologous end joining = disruption in reading frame of gene of interest
Homology directed repair = sequence of interest flanked by homology arms is knocked in
Describe CRISPR epigenetics
Broken Cas9 coupled to epigenetic modifiers can be used to study how specific modifications affect gene expression and DNA dynamics
Describe gene drive as a result of CRISPR gene editing
gene editing can be used to propagate genetic motif through generations
Cascade testing is a sequence of tests to diagnose a disease or process and starts by identifying what in the affected individual?
Describe three biochemical tests used in neonatal screening
Analytes, NZ assays, protein analysis
Describe 4 cytogenetic tests used in neonatal screening
karyotyping, FISH, CHIPS(microarray), CFF DNA (cell free fetal)
What is the limitation in sequence analysis testing?
Pt may have allelic variant of unknown significance --> until significance is determined, no meaningful risk info can be provided