DNA MICROARRAY Flashcards
STUDY OF HEREDITY
GENETICS
It focuses primarily on the likelihood of _____________.
Genetics - developing cancer
tests find ______, not diseases.
genetics - mutations
STUDY OF GENES, HOW THEY INTERACT AND EXPRESSED AS A WHOLE
genomics
If genetics is the study of ______, genomics is the study of _____ and how they interact, how they are passed on from generation to generation, and how they are expressed as a whole.
heredity, genes
Genetics and genomics are _________ that focuses on cancer itself and can help determine:
profiling tools
- prognosis (how aggressive)
- prediction (what is likely the benefit)
It is an international scientific research project.
HUMAN GENOMICS PROJECT
HUMAN GENOMICS PROJECT
→ Determining the _______.
→ ldentifying and mapping all of the ______.
sequence of human DNA, human genes
HUMAN GENOMICS PROJECT
Key Findings:
There are ________ genes in a human
being
approximately 20,500
They are the basic physical and functional unit of heredity
GENES
They are a part of a DNA.
GENES
Some genes act as instructions to make molecules called _____
proteins
Each human cell contains ___ chromosomes, and each chromosome contains _____ genes (based on the findings of the Human Genomic Project)
46
20,500
2 TYPES OF GENES
Structural and Regulatory Genese
A gene that codes for any RNA or protein product other than a regulatory factor.
structural genes
Encode for proteins or RNA
structural genes
Encode for mRNAs, rRNAS, and tRNAS.
structural genes
Gene products have either structural or functional importance
structural genes
A gene involved in controlling the expression of one or more other genes.
regulatory genes
Encode for transcription factors or regulatory RNA
regulatory genes
Regulate the expression of structural genes
• Regulatory genes process the ________.
• Ensures that the appropriate genes are __________
regulatory genes
- turning on and off of the gene
- expressed at the proper time.
EXAMPLES FOR STRUCTURAL GENES
Lac Z,A,Y genes of lac operon, actin gene
EXAMPLES OF REGULATORY GENES
Lac I gene, CAP gene
WHOLE DNA GENE 2 PARTS
- Intron
2. Exon
portion of a gene that does not code for amino acids/ proteins
intron
- part of thw whole gene that are just intermissions
- They are just there for the whole DNA to have time in forming a final mature RNA.
intron
portion of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing.
Exons
occurs only in a particular type of cell or
tissue where only a subset of an organism’s DNA will be expressed as mRNAs at any given time.
gene expression
The gene expression is focused on the _________ (________) where the 20,500 genes are activated simultaneously, and these genes are only activated when they are needed.
Central Dogma of Life, DNA -> RNA -> protein
The unique pattern of gene expression for a given cell or tissue is referred to as its __________.
*each gene has a unique pattern
molecular signature
If gene __ is activated (after the gene expression), it
means the patient has cancer. (Indication of cancer)
gene B
Why is gene expression important in any
malignancy/disease?
The use of gene expression profiling and development of gene biomarkers/signatures for cancer allows for the diagnosis, progression and aggressiveness analyses, prognosis, and prediction of therapeutic treatment.
MAIN TYPES OF GENE EXPRESSION ASSAYS
- Serial Analysis of Gene Expression (SAGE)
- Short Oligonucleotide Arrays SOA (Affymetrix)
- Long Oligonucleotide Arrays LOA (Agilent Inkjet)
- Fiber Optic Arrays FOA (Ilumina)
- cDNA Arrays (Brown/ Botstein)
It does not use the whole sequence of genes; it only
uses a small part of the gene.
Short Oligonucleotide Arrays (Affymetrix)
Father of Microarray Technology
Mark Schena
The slide cDNA microarray which was discovered by
Brown and Botstein.
cDNA array
Each slide contains multiple spots, and each spot
contains the DNA clone
cDNA array
The sample used in cDNA array is usually _______
PCR generated
Developed a scanner for reading the output
Steve Fooder
Developed a quantitative way of monitoring Gene Expression Patterns with complementary DNA Microarray.
Patrick Brown and Mark Schena
DNA Microarray is based on the ___________
Principle of Southern Blotting in the year 1975
The concept of DNA Microarray began in the ______.
mid-1980s
DNA microarray is also known as
DNA chip or Biochip
It is a technology that can sequence thousands of genes at a time in order to quantify the expression of the said genes
DNA Microarray
The bonding of probes and targets is referred to as
“HYBRIDIZATION”.
This is the DNA of a sample of interest which
is compared to a reference DNA or ‘control DNA’.
Target DNA
The gene that is placed into the hole in the
glass slide.
probe
________ in general, are a snapshot that captures the
activity pattern of thousands of genes at once.
microarray
a collection of features spatially arranged in a twodimensional grid, arranged in columns and rows.
the array/slide
The slide consists of a thousand to hundreds of thousands
of spots per square inch
Array /slide
Each spot holds ______ of copies of a DNA sequence from one gene
millions
3 MAIN TYPES OF DNA MICROARRAY
- Spotted array
- In-situ array
- Self-assembled array
→ These arrays use a poly-lysine coated microscope slide.
SPOTTED ARRAY
A robotic arm dips a small tip into DNA and then onto the glass surface to fluorescently label the sample
Spotted array
The probes need to be synthesized prior to chip preparation
spotted array
First arrays to be created
Spotted arrays
it is the most straightforward to use and relatively inexpensive.
spotted array
Allows the user to check the purity and quantity of the DNA or cDNA to be used
spotted array
can be time-consuming as the DNA or RNA have to be extracted, converted into cDNA (in the case of an RNA), and then purified.
Spotted array
These arrays use light and photolithography to create
probes on chips.
In-Situ Array
These probes are oligonucleotides that match down sequences of targeted DNA
IN-SITU ARRAY
These arrays are faster than spotted arrays and do not need cDNA handling; it only requires the target sequence to be known.
In-Situ Array
- have higher sensitivity
- are costly and require specialized machinery to synthesize many arrays themselves
In-Situ Array
These arrays are fiber optics.
Self-assembled array
DNA is assembled on small polystyrene beads.
SELF-ASSEMBLED ARRAY
Some versions contain wells already pre-made in a glass to hold the beads.
SELF-ASSEMBLED ARRAY
The beads are encoded with different combinations of fluorophore so that the randomly assembled oligos are identified by their specific combinations.
SELF-ASSEMBLED ARRAY
2 COMMON METHODS OF DNA MICROARRAY
- Glass Slide cDNA microarray
2. DNA chip (oligonucleotide) Microarray
→ Conventional method.
Glass Slide cDNA Microarray
First type of DNA Microarray technology developed.
Glass slide cDNA Microarray
Invented by __________ & his colleagues at Stanford University.
Glass slide cDNA microarray- Patrick Brown
Uses a robotic device which deposits (spots) a nanoliter sample of DNA onto a coated microscopic glass slide (Spot size: 50-150 micrometer in diameter).
Glass slide cDNA microarray
The slide is usually made commercially and is made of glass/silicon/nylon.
Glass slide cDNA microarray
Each slide contains thousands of spots, and each spot contains a probe for a different gene
Glass slide cDNA microarray
→ Probes can attach to the slide by robotic means.
Glass slide cDNA microarray
ADVANTAGES:
• Relatively affordable with a ____ cost.
• It requires _______.
• Prior knowledge of sequence is _______.
• Good choice for ______ discovery.
• Necessary clone sets are available for numerous
organisms (_______)
Glass slide cDNA microarray
- lower
- no specialized equipment
- not necessary
- new gene
- humans, mice, rats
DISADVANTAGES
• Requires ______ for synthesizing & purifying DNA solutions before microarray fabrication.
• _______ of probe (Slide DNA) as compared to oligonucleotides array.
• _________ may occur due to common sequences from the same gene family
Glass slide cDNA microarray
- intensive labor
- Low density
- cross hybridization
______ are high-precision robots with metal pins that dip into DNA solution and taps down on the glass
slide.
Microarray spotters
Glass slides cDNA microarrays uses ______
• DNA spotting done by _____ arm using multiple pins.
• DNA in a ____ plate.
• DNA is usually _______.
• ______ can also be spotted.
spotting arrays
- robotic
- microtiter
- PCR amplified
- oligonucleotides
The process is done by prefabricating or synthesizing single-stranded oligo.
DNA Chip (Oligonucleotide) Microarray
Since oligos are usually ____, the density of these arrays is much higher.
short - DNA Chip (oligonucleotide) microarray
DNA Chip (Oligonucleotide) Microarray is traditionally called
‘Gene Chips’ (Gene Chip Array) by
commercial suppliers, such as Affymetrix, Agilent
ADVANTAGES:
• Fast
• Specific
• Reproducible
DNA chip microarray
DISADVANTAGES
• In-situ oligonucleotide array formats tend to have _______ specialized equipment to carry out hybridization, staining of label, washing, and quantitation
process.
• Short sequences (oligonucleotides 10-15 base pair only) used on the array have __________ compared with glass cDNA microarrays.
DNA chip (oligonucleotide) Microarray expensive, decreased sensitivity/binding
DNA MICROARRAY (Procedure)
- Collection of samples
- Isolation of mRNA
- Creation of Labeled DNA
- Hybridization
- Microarray scanner
- Analyzation of Data
COLLECTION OF SAMPLES
Samples which can be collected:
a. peripheral blood
b. aspirate fluid
c. swab sample
d. lavages
- any cells that have DNA can act as sample
ISOLATION OF mRNA
- Extract the RNA from the samples. Using either a column or a solvent such as ________
- After isolating the RNA, we need to isolate the ____ from the rRNA and tRNA. The mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind with the mRNA.
- Rinse with _____ to release the mRNA from the beads. In turn, the buffer disrupts the Ph, disrupting hybrid bonds
phenol chloroform, mRNA, buffer
CREATION OF LABELLED DNA
• Add a labeling mix to the RNA. The labelling mix contains poly-T primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides
- Add Cyanine-3 (______) to the healthy cells.
- Add Cyanine-5 (______) to the cancerous cells.
fluoresces green, fluoresces red