1. Part 3 Gene Technology & Medicine

2. 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

3. PCR 3 Main Stages Denaturation Annealing Elongation Heat & Denature
Cool & Add Primer
65*C
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

4. PCR Process Denature DNA using heat Cool Annealing Elongation
Separates DNA into 2 single strands
Bases exposed
Cool
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
Elongation
DNA polymerase (Taq polymerase) used to build new strands of DNA against the exposed bases using free nucleotides
Repeat procedure

5. Benefits of Taq Polymerase
Not destroyed during high- temperature denaturation process
Does not need to be replaced for each cycle of PCR
High Optimum Temperature
Means that temperature for elongation stage does not need to be dropped below that needed for annealing stage

6. 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

7. Terms to Know Probe cDNA VNTRs
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

8. VNTRs Variable, repeated segments of human DNA
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

10. 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

11. 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

12. 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

13. 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

15. 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

16. 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

17. Why Sequence? Sequencing Genomes: Sequencing Amino Acids
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

18. 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

19. 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