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Flashcards in Week FO, FO SHO Deck (136)
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31

Describe microarray hybridization technique

Glass slides with probes that identify each gene, fluorescently labeled

32

DNA cloning uses cloning vectors and PCR. What two enzymes are required?

Restriction enzymes and DNA ligase

33

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)

34

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

35

Describe temperature steps for PCR

1) Denaturation (94C) 2) Annealing (depends on primer) 3) Extension (72C)

36

Real time PCR uses fluorescent probes/dyes to detect and quantify ____ in real time

DNA, if reverse transcriptase used, can quantify mRNA

37

Which of the following is not a use of PCR? a) genetic testing b) clone gDNA c) forensic/identity screens d) identify translocation

d)

38

What is analyzed in Western blot and how?

proteins, travel based on molecular weight

39

Regulated gene expression uses a ______________ to turn genes on and off

genetic switch

40

miRNA and siRNA both cleaved to mature form by _______

dicer enzyme

41

miRNA is _______ expressed while siRNA is ________ expressed

endogenously, exogenously

42

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

43

What are the four requirements for DNA sequencing?

free 3'OH group, DNA polymerase, primer, template

44

Describe Sanger sequencing

ddNTPs incorporated to initiation chain termination - no free 3' OH end

45

How would this gel (result of sequencing) be read?

From bottom up (ATGCT etc)

46

Describe an antisense molecule

Bind to specific complimentary sequences and inhibit gene expression

47

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

48

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)

49

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

50

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. 

51

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)

52

Pronuclei injection can also be used to generate transgenic animals. Describe. 

Desired gene injected into fertilized egg --> egg implanted

53

Describe the two components of CRISPR gene editing

Cas9 protein - molecular scissors

guide RNA - guides cas9

54

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

55

Describe CRISPR epigenetics

Broken Cas9 coupled to epigenetic modifiers can be used to study how specific modifications affect gene expression and DNA dynamics

56

Describe gene drive as a result of CRISPR gene editing 

gene editing can be used to propagate genetic motif through generations

57

Cascade testing is a sequence of tests to diagnose a disease or process and starts by identifying what in the affected individual?

mutation

58

Describe three biochemical tests used in neonatal screening

Analytes, NZ assays, protein analysis

59

Describe 4 cytogenetic tests used in neonatal screening

karyotyping, FISH, CHIPS(microarray), CFF DNA (cell free fetal)

60

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