DNA Tools and Biotechnology Flashcards
Biotechnology
the manipulation of organisms or their components to make useful products
Genetic Engineering
the direct manipulation of genes for practical purposes
Uses of Biotechnology (4)
- Medical processes
- Industry
- Agriculture
- Genetic engineering
may involve use of recombinant proteins or genetically modified organisms
2 Methods of DNA Sequencing
- dideoxyribonucleotide chain termination sequencing
- next generation sequencing
Gene Expression
the conversion of the information encoded in a gene first into mRNA and then into a protein
Recombinant DNA
- Genetically engineered DNA made by combining two or more sequences that would not normally occur together
- Example: as fragments of DNA from two different organisms
Recombinant Proteins
those that have been produced via recombinant DNA
Clone
recombinant DNA is often used to do this to genes
Plasmid
- A circular piece of bacterial DNA that normally exists separately from the bacterial chromosome and can replicate independently of the bacterial chromosome
General Steps of Recombinant Proteins (4)
- Isolation: genes of interest removed from organism
- Amplification using PCR
- Insertion into vector (plasmid)
- Transformation into host
Endonucleases
- enzymes that cut DNA at specific nucleotide sequences called restriction sites
- “molecular scissors”
- they cut the phosphodiester backbone
What is a Vector? (2)
- “Vehicle”
- DNA molecule, like a plasmid or virus, used to deliver a specific DNA segment into a host cell for cloning or genetic engineering
2 Types of Vectors
- Virus
- plasmid
How Restriction Enzymes Can Be Used To Create Recombinant DNA (2)
- restriction enzyme (Endonuclease) cuts sugar-phosphate backbones of DNA at specific sequences
- “Sticky” ends are created to facilitate joining of different DNA fragments by DNA ligase
Transfection
the process of introducing foreign nucleic acids (DNA or RNA) into eukaryotic cells using non-viral methods.
Transformation
the process by which bacteria take up the new gene and expresses the protein from
Transduction
The transfer of genetic material (DNA) from one bacterium to another via a bacterial virus, also known as a bacteriophage.
Transgenic Organisms
- Organisms whose genomes have been altered through introduction of a foreign gene
- AKA genetically modified organisms
Polymerase Chain Reaction
- (PCR) a technique used to amplify DNA
- Rapid and relatively inexpensive technique to amplify DNA and determine presence/absence
cDNA synthesis
- Complementary DNA
- Created from an RNA template through reverse transcription
- Very stable whereas mRNA is generally not so stable
Real time PCR
- widely used to determine transcript abundance
- rapid
- accurate
- more expensive
- combines amplification and detection
Next Gen Sequencing
- used for DNA or RNA sequencing (RNAseq)
- ultra high throughput -
ultra high speed and scalability - costly
What can RNAseq be used for? (3)
- Quantify gene expression
- Identify novel transcripts
- Study alternative splicing
Gene Cloning
- method of amplification
- can be stored as a plasmid or in bacteria and then transformed into another organism or to produce products of interest
- relatively rapid and inexpensive
Primers
short DNA segments that are complementary to the 3’ ends of the DNA to be amplified and provide a starting point for DNA polymerase
Basic Process of PCR
- amplify DNA carried out with thermal cycler
- cycle consists of 3 stages:
1. denaturing
2. annealing
3. extending
Sequence Variant {Alteration}
any sequence change (compared with reference sequence)
Polymorphism
The occurence of variation in a specific DNA sequence in a population
Single Nucleotide Polymorphism {SNP}
a DNA sequence variation occurring when a single nucleotide in the genome differs between members of a species or paired chromosome in an individual
Variable Number Tandem Repeats {VNTRs}
- Repetition of a short nucleotide sequence
- Number of repeats varying between individuals
Genetic Profiling and uses (3)
- Analysis of an individuals DNA to allow fofr comparison and identification.
- AKA “fingerprinting”
- Forensics, paternity testing, identification
Steps In Genetic Profiling
- isolate and amplify DNA with PCR
- perform restriction digest using restriction enzymes
- run the restriction fragments on gel electrophoresis
- stain the gel and analyse the pattern of DNA to identify an individual
Restriction Fragment Length Polymorphisms {RFLPs}
- Detects differences in homologous DNA sequences by observing variations in the lengths of DNA fragments produced after digestion with restriction enzymes
Gel Electrophoresis
the size based separation of molecules (such as DNA) when an electric current is applied to a gel
Uses of Genetic Profiling
- criminal investigations
- paternity testing
- matching recipients with organ donors (HLA typing)
- identifying microorganisms or detect sequences of RNA
Nucleic Acid Probes
Short, synthetic sequences of DNA or RNA used to detect complementary DNA or RNA sequences in a sample
Microarrays {Biochips}
- Solid chips with short sections of DNA or RNA attached to the surface.
- Used to test for a DNA of interest or to examine mRNA expression levels (an indicator of what proteins are being made)
3 Ways to Determine a Genes Function
- compare a gene’s sequence and function to that of a known gene in another species
- Mutagenesis
- RNA interference
Human Genome Project
the goal of this was to determine the location (and sequence) of genes on human chromosomes
Stem Cells
- Undifferentiated cells that are able to produce many different kinds of cells and tissues.
- Their only function is to produce daughter cells
Embryonic cells
- totipotent or pluripotent
- come from blastocyst stage of embryo
Adult cells
- multipotent
- undifferentiated cells present in differentiated tissues such as blood and skin
Induced Pluripotent Stem Cells {iPS}
cells that have been induced into a state of pluripotency through the introduction of genes from outside their genome
Somatic Cell Nuclear Transfer
cloning designed as therapy for a disease
Possible Use of Therapeutic Cloning
replace tissues damaged by spinal cord injury or burns
Gene Therapy
- a technique for correcting defective genes responsible for disease development
- replaces defective alleles with normal alleles
- successfully used in humans
- not currently approved by FDA
Carriers of DNA For Gene Therapy (3)
- viral vector
- lipoplexes
- naked DNA
Lipoplexes
lipid spheres encapsulating genes that are directly injected into the patient
Naked DNA
DNA that is not carried by any vector but is injected directly into a patient’s muscle
Problem to overcome for Gene Therapy
an immune reaction by the recipient may kill the viral vector
Reproductive in vivo cloning is
making an exact copy of an organism
Pros & Cons of Genetic Engineering
- curing diseases
- big companies will shut down small farms or lack of genetic diversity
Pharmacogenomics
the study of how an individual’s genes affect his/her body’s response to drugs
