Week FO, FO SHO Flashcards Preview

B1 > Week FO, FO SHO > Flashcards

Flashcards in Week FO, FO SHO Deck (136)
Loading flashcards...

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

genetic switch


miRNA and siRNA both cleaved to mature form by _______

dicer enzyme


miRNA is _______ expressed while siRNA is ________ expressed

endogenously, exogenously


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