Unit II- Molecular Technology Flashcards
1
Q
What is Molecular Biology
A
- recombinant DNA technology or molecular genetics
- a collection of methods used to isolate, manupulate, amplify, re-organize, and analyze genes at the molecular level
- methods that have revolutionized and will continue to revolutionize research, diagnostics, and practice of medicine
- made possible by learning: the structure of DNA, restriction and modification of bacteria, DNA sequencing, DNA uptake by bacteria, bacterial plasmids and cloning, therophilic bacteria, automation, minitiarization, deep sequencing
2
Q
Nucleic Acid Hybridization
A
- inherent feature of DNA/RNA: double stranded complementarity
- allows one to “search” for homologous sequences in complex mixtures and/or to discriminate between related sequences
3
Q
Restriction Enzymes
A
- part of the bacterial defense system called restriction and modification
- usually cuts dsDNA in palindromic sequences
- can cut to leave staggered ends (most useful) either with 5’ and 3’ overhands
- staggered ends can be efficiently ligated to complementary ends with T4 DNA Ligase
- can serve as landmarks on DNA sequences and therefore can be used for linkage analysis (RFLPs)
- think EcoRI
4
Q
Restriction Maps
A
- formed the basis for the 1st molecular method to perform genetic linkage analysis called RFLPs
- shows where all the restriction enzymes cut
5
Q
Sizing of nucleic acids: Gel Electrophoresis
A
- nucleic acid is separated by size through a molecular sieve (gel)
- voltage is used to move the negatively charged nucleic acids toward the anode (positive_
- two gel choices: agarose-large, acrylamide-small
- detection: Fluorescent dyes (EtBr), Radioactivity-film or phosphoimager screen, probes
6
Q
Labeling DNA to make “probes”
A
- isolate fragment to be labeled
- prime a DNA synthesis reaction with homologous oligos
- synthesize complementary strands with a DNA polymerase in the presence of radioactively labeled (32P) nucleotide precursors
7
Q
Southern Blotting
A
- separate DNA by size using gel electrophoresis
- make a paper copy of the gel: use voltage to transfer the DNA onto nitrocellulose paper, HIGH pH and/or salt denature the DNA
- hybridize a 32P-labeled DNA “probe” to the paper
- wash non-specific probe by binding off of the paper
- expose the paper to X-ray film or a phospho-imager screen
8
Q
Cloning
A
- isolate DNA fragment and vector cut to leave completementary “sticky” ends
- ligate (splice) the fragment into the vector
- transform the chimearic molecules into a host (bacteria)
- screen for the clones with the insert
9
Q
Vectors
A
- vehicles used to move, manipulate and amplify cloned genes/fragments of genes
- they are human altered versions of naturally occuring DNA elements used to transfer genetic information
- they vary in the amount of recombinant DNA they can be tricked into carrying and the hosts that can maintain them
10
Q
Bacterial plasmids
A
- based upon F1 plasmids
- small closed, dsDNA circles
- bacteria are the host, introduced by chemical transformation
- carry an origin of replication, an antibiotic resistance gene and a poly-cloning site (a short stretch of DNA that has many restriction enzyme cut sites)
- can only accept up to 15 kb of recombinant DNA
11
Q
Bacteriophage Lambda
A
- a bacterial virus with a 45 kb linear dsDNA genome
- during infection viral genes hijack the bacterium to produce millions of viral particles
- bacterial lysis spills the viral particles into the growth medium
- recombinant DNA up to 20 kb can be inserted into the middle non-essential region of the viral genome
12
Q
Cosmids
A
- gutted versions of lambda all but the ends have been removed
- can clone up to 45 kb of recombinant DNA
- recombinant genome is packaged into viral particles in a helper strain: Cos ends direct replication and packaging
13
Q
Bacterial Artificial Chromosomes (BACs)
A
- dsDNA closed circles (plasmids) that are maintained in E. coli as if they were the cell’s chromosome (low copy #)
- can accept 100-300 kb of recombinant DNA
14
Q
Yeast Artificial Chromosome (YACs)
A
- linear dsDNA recombinant molecules maintained in yeast as a chromosome
- telomeres on ends, centromere in middle and origins of of replication insure stable maintenance
- can accept 100-2,000 kb recombinant DNA
- not as good because they cross-over more
15
Q
Libraries
A
- a large collection of random inserts for a genome of interest in a cloning vector
- analogous to the Library of Congress after a large earthquake (all mixed up)
- used to “fish out” the gene or genes you are interested in studying
- ideally all parts of the genome are represented as overlapping fragments
- two types: gemonic and cDNA
16
Q
cDNA library
A
- purify mRNA from cells/tissue of interest
- affinity purify on an oligo-dT coupled column to isolate poly(A) mRNA
- prime with oligo-dT and make complementary DNA strands with Reverse Transcriptase
- Degrade the RNA strand
- Use DNA polymerase to make the second DNA strand
- ligate into a cloning vector (usually a bacterial plasmid)
- representatives in the library are only expressed genes with introns removed
17
Q
Screening a library for a gene
A
- there is a petri disk with colonies of bacteria containing recombinant plasmids
- a piece of absorbant paper is put on, and the paper is peeled from dish to produce replica of colonies
- lyse bacteria and denature DNA with alkali
- radioactively labeled DNA probe is added
- bacteria incubated with probe and washed
- colonies containing plasmid of interest were exposed to photographic film
18
Q
PCR
A
- if you know the sequence of a region of nucleic acid (RNA or DNA), PCR allows you to amplify the sequence from complex mixtures of nucleic acids even if the copies of that sequence are inabundant
- requires 2 primers, dNTPs, starting sample that contains the first template, a thermophilic polymerase
- bionomial expansion: amount of DNA made= # of starting templates x 2^#cycles. Product analyzed by gel electrophoresis
19
Q
Analysis of PCR products
A
- use gel electrophoresis to answer the following:
- did it work? Did i get product?
- what is the size of the product?
- how much product did I get?
How much template did I start with- Real Time PCR- measure the rate of product production
20
Q
Multi-Plex PCR
A
- detection of exon deletions in the Dystrophin gene-
- looking for a lot of gene sequences in one gel
21
Q
Test for HIV
A
- Reverse Transcriptase PCR
- blood sample from the infected person
- removed cells by centrifugation
- extract viral genome
- Reverse transcriptase/ PCR amplification
- run at gel, also use control from non-infected person which should have no bands
22
Q
PCR and cloning
A
-used to make many copies of protein of interest for example actin
23
Q
Analysis of Gene Expression
A
- answers- does the patients tissue express a particular gene?
- is the gene product of the correct size, expressed at the proper abundance, in the correct cells, and in the correct location in those cells
- size and expression of the mRNA: Northern Blot or Reverse Transcriptase PCR
- whole genome “transcriptome” analysis: DNA microarrays, tiling arrays, and RNA sequencing
- size and expression level of the protein: Western Blot
- proper cellular expression and localization: Immunofluorescence Microscopy or live cell imaging using GFP fusion proteins
24
Q
Northern Blots
A
- detects mRNA
- very similar to Southern Blots except purified mRNA (polyA+) is separated on the gel
- answers: is the mRNA expressed, ~expression level and proper length
25
Denaturing Protein Gel Electrophoresis
- protein is boiled in the presence of sodium dodecyl sulfate (SDS) and +/- reducing agent (mercaptoethanol).
- creates a uniform ratio of negative charge to size
- separation in polyacrylamide is purely by size
26
Non-denaturing/ native protein gel electophoresis
- can be used to separate proteins based on size/ shape and charge at a particular pH
- sample is not heated and no SDS or reducing agent is included
- it can be difficult to predict how any given protein will migrate in the non-denaturing gel but can be used to compare variants of the same protein: for example it can be used to compare proteins with mutations in charged amino acids such as certain mutations in hemoglobin that cause sickle cell disease
27
Isoelectric Focusing (IEF)
- proteins migrate in a stable pH gradient constructed in a polyacrylamide gel
- proteins stop migrating once they reach their isoelectric point (pI): the pH at which they carry no net charge
- two-dimensional gel electrophoresis combines IEF and standard denaturing gel electrophoresis. Very useful to analyze complex mixtures of proteins (proteomics)
28
Two-Dimentional protein gel electrophoresis- "Proteomics"
-by coupling two-dimensional gel electrophoresis with mass-spectrometry, we can now identify all of the proteins expressed in a cell or tissue and how they are post-translationally modified
29
The use of antibodies to study proteins
- antibodies are raised in animals by injecting them with the antigen of interest- "primary antibody"
- "secondary antibodies" are antibodies raised against the contstant regions of antibodies from a different animal species- usually labeled by fluorescent or other tag
30
Analysis of Dystrophin from Patients with Muscular Dystrophy
- isolate protein from a muscle biopsy
- separate proteins by gel electrophoresis
- transfer proteins to nitrocellulose paper
- blot with a primary antibody against the dystrophin protein
- blot with a labeled secondary antibody and detect
- answers:is the protein present, ~expression level, and size
31
Immuno-Fluorescence Microscopy Analysis of Dystrophin in muscle biopsy
- is the protein expressed in the correct cells and in the correct location in those cells? NO
- isolate a thin tissue section from muscle biopsy
- fix/preserve the sample
- blot with an antibody against dystrophin
- blot with a fluorescently tagged secondary antibody
- visualize anti-body/antigen localization on a fluorescence microscope
32
Other microscopy-3D
-three-dimensional fluorscence microscopy by deconvolution
33
Global Analysis of Gene expression
- miniaturized nucleic acid hybridization/detection
- robots spot DNA probes (one per gene) to glass slides (DNA microarray)
- microscope reads and quantifies the amount of RNA that is hybridized
34
Two color scheme
-mRNA from sample i (reference sample)
and then reverse transcribe with fluorescent nucleotide Cy3
-mRNA from sample 2 (experimental sample) and then reverse transcribe with fluorescent Cy5
-mix the complex probes together and hybridize overnight
-scan and determine fluorescence intensities at each spot
35
What do micro-array expression experiments tell us?
- the relative expression levels of all genes in the genome under specific conditions
- how this expression pattern changes when the system is perturbed, in different tissues, during development, in disease states etc etc
- identifies groups of coordinately regulated genes that may be functionally related
- provides a high resolution diagnostic tool to type a tissue, tumor, disease state, physiological condition etc etc
36
Cluster Analysis
- compare all expression patterns to each other
- join patterns that are the most similar
- compare joined patterns to all other unjoined patterns
- go to step 2, and repeat until all patterns are joined
37
Cluster Analysis of serum fed fibroblasts
- data is lumped together (clustered) based on extent of shared behavior in multiple experiments
- starved fibroblasts were fed serum and mRNA was isolated over time and compare to RNA from serum starved control cells
- red are genes induced relative to the control and green are genes repressed- the wound healing genes are turned on, the cell cycle genes turned on at about 24 hours
38
Micro-Array Analysis of Lung Cancer
- 67 human lung tumors
- expression of 24,000 genes analyzed and compared to normal lung tissue
- tumors clustered based on shared changes in gene expression
- tumors fell into pathologically defined groups, except...
- pathologically indistinguishable adenocarcinomas clustered into three groups that shared patient outcomes
39
Other uses of micro-arrays
- genotyping SNP chips
- whole genome mapping of protein binding sites: ChIP/chip
- tiling arrays: several million feature arrays, mutation detection, transcript mapping
- protein chips
- pathogen detection
- miniaturization coupled with automation is driving tremendous innovation in research and diagnostics
40
Next generation sequencing
- whole genome sequencing and assembly for whole genome genotyping
- RNA-seq or Whole Transciptome Shotgun Sequencing- transcript abundance, mutations in transcribed genes, gene fusions
41
RNA Interference (RNAi)
- discovered in C. elegans
- introduction of dsRNA corresponding to a cellular mRNA will induce degradation of the mRNA and down regulation
- dicer endonuclease and helicase processes the dsRNA into 23 nt pieces
- processed siRNA hybridizes to the mRNA
- dicer cleaves the mRNA and nucleases complete the degradation
- the RNAi reaction is amplified and can be quite persistant
42
Problems with potential clinical applications of RNAi
- delivery-getting it to the right tissue and getting it into cells
- long dsRNA induces a powerful interferon response-shRNAs tend to do so less
- sometimes there is poor specificity of the dsRNA for its intended target
43
Advantages and anticipated uses of RNAi-like approaches in humans
- the main advantage is that a natural system of gene expression is being harnessed. So far ~200 natural siRNAs have been identified in the human genome
- some dsRNA viruses have defenses against RNAi: flock house, influenza, and vaccinia virus. Their defense systems would make good drug targets
- suppression of dominant alleles or diseases due to inappropriate over-expression
- can use lentiviral deliverty of an shRNA into Mammalian cells
44
"Morpholinos"
- short single stranded oligos complementary to a transcript you want to knock down
- inhibit expression by blocking progress of the ribosome/translation or by blocking a splice site and inhibiting splicing
45
Currently in development
- treatment for macular degeneration by down regulation of VEGF in the retina
- treatment of Hepatitus B and C infections
- siRNA has shown some promise in treating infections by HIV, syncytial virus, influenca virus and polio
- early promise for silencing the dominant allele that causes amyotropic lateral sclerosis
46
Protein Localization in Living Cells: Green Fluorescent Protein
- naturally fluorescent protein from the jellyfish Aquoria victoria
- the GFP gene can be introduced into any cell and they will produce an active fluorescent GFP protein
- can be fused to any gene of interest and used to study the expression and/or localization of the gene product in living cells
