Exam 4 (Topic 16) Flashcards
Enzymes that cleave phosphodiester bonds
Restriction Endonucleases
Enzyme that is made by bacteria only to protect from foreign DNA
Restriction Endonucleases
Enzyme that only cut DNA at specific nucleotide sequences by chance
Restriction Endonucleases
Puts cut DNA back together
DNA ligase
In an electric field DNA moves from the negative electrode to the positive electrode and separates based on size
Agarose gel electrophoresis
DNA can be stained with a fluorescent dye to visualize with UV light. DNA bands can then be cut out of gel and isolated.
Agarose Gel Electrophoresis
The isolation and amplification of a specific region of DNA
DNA Cloning
DNA fragments cut by Restriction endonucleases and put back together with DNA ligase
Recombinant DNA
Specialized circular DNA molecules used to transform bacteria
DNA Plasmids (recombinant)
Multiple Cloning Site (MCS)
Short segment of DNA that allow biologists to insert a piece or pieces of DNA into the region
Origin of replication
Tells bacteria to replicate
Selection Gene
Code for drug resistance
Can be amplified and purified in bacteria
Recombinant DNA
The introduction of DNA into bacteria
Transformation
Amplifying DNA fragment in vitro (without the use of bacteria)
Polymerase Chain Reaction (PCR)
Can make billions of copies of a segment of DNA
Polymerase Chain Reaction (PCR)
Extremely sensitive, can detect a single template in a sample
Polymerase Chain Reaction (PCR)
For PCR you need the following materials:
Template DNA, Forward and Reverse primers that are complementary to the template DNA, Nucleotides (dATP, dGTP, dCTP, dTTP) to generate new DNA, DNA polymerase
PCR Uses
Test for viral infection in a blood sample
Isolate viral genome (usually in the case of HIV)
Forensic medicine
A DNA copy of RNA
cDNA
How do you create cDNA
Reverse Transcriptase
Amplifies the viral genome and detects the amplified DNA on a gel
PCR
Regions of DNA on all chromosomes that is highly variable and unique to each individual
Short Tandem Repeats(STRs) in DNA Fingerprint
In order to study the function of a gene we must know
its DNA sequence
Dideoxy DNA sequencing requires
Deoxyribonucleotide (dNTP) Dideoxyribonucleotide Triphosphates (ddNTP)--smalle quantity compared to normale dNTPs 1 Primer to start the PCR reaction Template DNA to be sequenced DNA polymerase
How often is dideoxyribonucleotides incorporated in new DNA
Rarely
Are missing the 3’-OH and terminate replication of that strand
Dideoxyribonucleotides during Chain Termination
Old fashion DNA sequencing
Do 4 PCR reaction where each reaction contains a single dideoxyribonucleotide in small amounts and regular deoxyribonucleotides. Generates lots of DNA fragments each varying in size where the the dideoxyribonucleotide was inserted. Run aragrose gel of each PCR reaction
DNA sequencing today
Each dideoxyribonucleotide is labeled with a fluorophore (4 bases = 4 colors). We can now measure the fragment size and using a computer that generates a color coded DNA sequence
The appearance or behavior an individual organism or protein (red eye vs. white eye)
Phenotype
The gene responsible for a given phenotype
Genotype
Gene function can be determined by identifying
Mutant Phenotypes
We can alter the genotype with the use of
Recombinant DNA technology
We can replace a normal gene with a defective gene
Gene Replacement
We can remove a gene completely from an organism
Gene Knockout
We can add a new gene to an organism
Gene addition
Any organism who has had its genome altered in any way
Transgenic Organism
Injected C. elegans with dsRNA oligonucleotide that can base pair with mRNA sequence would inhibit protein production.
1998: Craig Mello and Andrew Fire
Craig Mello and Andrew Fire
Discovered RNA interference (RNAi) and miRNAs
The human genome codes for 400 miRNAs which regulate what?
At least 1/3 of all protein encoding genes
Transcribed in the nucleus and exported to the cytoplasm
miRNA
RNA Interference miRNA Process
- dsRNA binds to RISC complex
- Unwinds dsRNA and makes ssRNA
- ssRNA/RISC comples binds to complementary sequence on mRNA
- ssRNA/RISC complex degrades mRNA
Generated from foreign dsRNA
siRNA
RNA Interference siRNA Process
- Cleaved by enzyme Dicer into small dsRNA (21 nucleotides)
- dsRNA binds to RISC complex which unwinds RNA and makes ssRNA, binds to complementary sequence on mRNA and degrades mRNA
Enzyme that cleaves dsRNA into small sdRNA
Dicer
RNAi in the lab
If you know the gene sequence, design siena complementary to the mRNA that will cause the mRNA to be degraded and the protein will no longer be made
hen the mRNA is degraded and the protein will no longer be made
Gene knockdown
Knocking down gene expression
Gene knockdown
To investigate protein fuction, RNAi can be used to…
decrease the amount of protein made in a cell (knockdown)
What is responsible for unwinding double stranded RNA and degrading complementary mRNA?
RISC Complex
