Download File

Ch. 19
Part 3
Gene Technology & Medicine
Polymerase Chain Reaction (PCR)
• Method for rapid production of a
large number of copies of a
specific DNA fragment
• DNA Amplification
• Coupled with Gel Electrophoresis
• Allows unlimited number of
copies of DNA to be reproduced
from one molecule
• Useful in forensics
• Make millions of copies of a DNA
fragment extracted from a small
piece of DNA left behind at a crime
scene
PCR
• 3 Main Stages
1. Denaturation
• Heat & Denature
• 95*C
2. Annealing
• Cool & Add Primer
• 65*C
3. Elongation
• DNA polymerase adds nucleotides
• 72*C
• Taq polymerase  1st stable DNA polymerase
used in PCR
• Isolated from thermophilic bacteria
• Enables enzyme to work in HEAT
PCR Process
1. Denature DNA using heat
• Separates DNA into 2 single strands
• Bases exposed
2. Cool
3. Annealing
• Attach primers to ends of single DNA
strand
• Primer:
• small length of DNA (20 base pairs long)
• Base sequence complementary to start of
part of DNA to be copied
• Attached to beginning of DNA fragment so
DNA polymerase has somewhere to attach
to
4. Elongation
• DNA polymerase (Taq polymerase) used
to build new strands of DNA against the
exposed bases using free nucleotides
5. Repeat procedure
Benefits of Taq Polymerase
1. Not destroyed during hightemperature denaturation
process
• Does not need to be replaced
for each cycle of PCR
2. High Optimum Temperature
• Means that temperature for
elongation stage does not need
to be dropped below that
needed for annealing stage
Microarrays
• commonly known as DNA chip or biochip
• collection of microscopic DNA spots
attached to a solid surface
• Allow for the study of a large # of genes in a
short period of time
• Increases information available to researchers
• DNA microarrays used to:
• measure the expression levels of large numbers
of genes simultaneously
• to genotype multiple regions of a genome
• Identify genes present in an organisms genome
• Find out which genes are expressed within cells
• Compare the genes present in two different
organisms
• To See which genes are being expressed in a
specific cell at any given time
• Analyze genomic DNA
Terms to Know
• Probe
• Short sequences of genetic information that match specific
complementary regions of DNA/RNA (such as VNTR regions)
• technique developed in the 1980s that involves a fragment of DNA
or RNA which has been labelled with a radioactive isotope or a
fluorescent marker
• Used to detect the presence of a particular sequence of bases in
another piece of DNA or RNA
• Come from known locations across chromosomes of involved
organisms
• 500 or more base pairs in length
• cDNA
• complementary DNA
• double-stranded DNA synthesized from a messenger RNA (mRNA)
template in a reaction catalyzed by the enzyme reverse
transcriptase
• often used to clone eukaryotic genes in prokaryotes
• VNTRs
• variable number tandem repeat
• a location in a genome where a short nucleotide sequence is
organized as a tandem repeat
• Can be found on many chromosomes
• Often show variations in length between individuals
VNTRs
• variable number tandem repeat
• Variable, repeated segments of
human DNA
• Each person’s set of VNTR is UNIQUE
• GOOD identifier
• Only identical twins share VNTRs
• Use restriction enzymes to cut DNA
into VNTR regions
• Separate using gel electrophoresis
• Compare movement:
• Long VNTR  do not move far
• Short VNTR  move quickly through gel
Structure of Microarrays
• Made on a small piece of glass/plastic
• 2 cm2
• Short lengths of single stranded DNA
attached to glass in a 2D pattern
• 10,000 positions per cm2
• Each position has MULTIPLE copies of
same DNA probe
• You can search database to find DNA
probes for a huge range of genes
• Once gene probes required for
microarray have been selected,
automated process applies probes to
positions on microarray
• Information gathered
Process of Using Microarrays to Compare DNA from 2 Different Species
• DNA collected from each species
• DNA cut into fragments
• DNA is denatured to yield single-strand fragments of
different lengths
• DNA labeled with fluorescent tags
• Green tag for species A
• Red tag for species B
• Labelled DNA is mixed together
• Labeled DNA is allowed to HYBRIDZE with probes on
microarray
• Any DNA that does NOT bind to probes on microarray is
washed off
• Microarray is inspected with UV light source
• Causes fluorescence
• Fluorescence = hybridization has occurred due to DNA
fragments being complementary to probes
Outcomes of Microarray Analysis
• Green and red fluorescent spots
• DNA from one species has only
hybridized with those specific
probes
• Yellow fluorescent spots
• Occurs when DNA from BOTH
species have hybridized with the
probes
• Means 2 species have same exact
sequence of DNA for that
fragment
• Same genes = common ancestor
• No color/fluorescence on a
specific position in microarray
• No DNA has hybridized with
probe
• Gene is NOT found in either
species
Detecting Genes Expressed in Cancer Cells
• Genes in cancer cells are different that genes in active,
non-cancerous cells
• Microarrays compare which genes are active by:
• Identifying genes being transcribed into mRNA
• Steps:
•
•
•
•
•
•
mRNA from 2 types of cells is collected
Reverse transcriptase used so that mRNA can make cDNA
PCR used to amplify cDNA
cDNA labelled with fluorescent tags
Labelled DNA is denatured to make single-stranded DNA
Single-stranded DNA allowed to hybridize with probes on
microarray
• Spots on microarray that fluorescent = genes that have been
transcribed by cell
• Intensity of light emitted indicate level of activity of each gene
• High intensity  many mRNA molecules present
• Low intensity  fewer mRNA molecules present
Results of
Microarray show
TWO things:
• Show which
genes are
active
• The level of
activity of
specific genes
in specific cells
Bioinformatics
• Collection, processing, and analysis of
biological information and data using
computer software
• Combines biological data with computer
technology and statistics
• Builds databases and allows links to be made
between them
• Computer technology facilitates the collection
and analysis of mass if information and allows
access via internet
• Gene Data generated from multiple
resources:
• DNA chips (microarrays)
• Gene sequencing
• Establishes sequences of base pairs in sections of
DNA
• Automated process
• Genomes of different species published
• Amino acid sequences
• Protein structure, shape, and function
• Primary structure
What is Sequencing?
• process of determining the precise order of nucleotides
within a DNA molecule
• It includes any method or technology that is used to
determine the order of the four bases—adenine,
guanine, cytosine, and thymine—in a strand of DNA
• Genome sequencing is often compared to "decoding"
• sequence is still very much in code
• a genome sequence is simply a very long string of letters in a
mysterious language
• Caenorhabditis elegans (C. elegans)
•
•
•
•
1st multicellular organism to have entire genome sequenced
Less than 1000 body cells (300 nerve cells)
Transparent
Model organism for studying genetics of organ development &
development of neurons
• Plasmodium
• Genome has been sequenced and is in database
• Information used to determine ways to control parasite
• Development of vaccines for malaria
Why Sequence?
• Sequencing Genomes:
• Enables comparisons
between genomes of
different organisms
• Sequences are matched
and similarities are
calculated
• Sequencing Amino Acids
• Compare sequences of
amino acids and proteins
• Compare structure of
proteins
• Close similarities =
common ancestry
Databases
•
•
•
•
•
Hold different types of information
DNA structures
Primary structures of proteins
Information is searchable
Examples:
• Ensembl
• Contains data on genomes of eukaryotic organisms
• UniProt
• Universal protein resource
• Holds info on primary sequences of proteins and
functions of many proteins
• BLAST
• Basic Local Alignment Search Tool
• Search tool
• Algorithm used to compare primary biological sequence
information
• Primary sequences of different proteins or nucleotide
sequences of genes
• Looks for similarities between sequences being studied
and what is in database
Using Microarrays and Bioinformatics
• Human development genes can
be found in the fruit fly (easy to
study)
• Microarrays find out when &
where genes are expressed during
development of fruit fly
• Databases  enable researchers
to access information about these
specific genes and the proteins
they code for
• Search databases for similar or
identical base sequence sin other
organisms
• Compare primary structures of
proteins
• Visualize 3D structures of proteins