Lecture 23: Technology Flashcards
Recombinant DNA: The Dawn of Molecular Biology
What is recombinant DNA?
DNA formed artificially by combining constituents from different organisms
Recombinant DNA: The Dawn of Molecular Biology
What is recombinant DNA created from?
Restriction endonuclease digested genomes, ligated into plasmids
Recombinant DNA: The Dawn of Molecular Biology
How is DNA cleaved?
DNA is cleaved at specific sequences by restriction endonucleases
- They often bind and cut the phosphodiester backbone at palindromic sequences
Recombinant DNA: The Dawn of Molecular Biology
What happens if restriction endonucleases cut the DNA and leave overhangs?
Sticky ends
Recombinant DNA: The Dawn of Molecular Biology
What happens when enzymes cut at the same site on both strands?
Blunt ends
Recombinant DNA: The Dawn of Molecular Biology
What is ligation?
Joining fragments together
Recombinant DNA: The Dawn of Molecular Biology
What are plasmids?
Very small circular DNA w few genes
- Selected for via ABX resistance markers
Recombinant DNA: The Dawn of Molecular Biology
Describe the relationship between DNA, endonucleases, DNA ligase, and plasmids
Endonucleases cut a piece of DNA
The fragment gets pasted into a plasmid and ligated with DNA ligase
Recombinant DNA: The Dawn of Molecular Biology
What is bacterial transformation?
Bacteria take up foreign DNA from environment
Recombinant DNA: The Dawn of Molecular Biology
What marker do plasmids contain?
They contain a selection markers
Recombinant DNA: The Dawn of Molecular Biology
What are selection markers?
A specific gene in a plasmid that allow for the identification and selection of cells that have successfully taken up the plasmid
Recombinant DNA: The Dawn of Molecular Biology
Describe the relationship between recombination DNA, a host cell and a foreign cell
Uptaken DNA can recombine with the host’s chromosomal DNA
- The host incoperates the new DNA into its cell machinery
- Can lead to genetic changes such as antibiotic resistance
Recombinant DNA: The Dawn of Molecular Biology
What genes are some selection markers?
Genes that confer antibiotic resistance
Recombinant DNA: The Dawn of Molecular Biology
What bacterial cells will grow into a medium containing the corresponding antibiotic?
Bacterial cells transformed with the
plasmid containing an antibiotic
resistance gene will grow in a medium
containing the corresponding antibiotic
Recombinant DNA: The Dawn of Molecular Biology
What cells will survive and grow in terms of antibiotic resistance?
Cells that have successfully taken ip the plasmid
PCR
What does PCR stand for?
Polymerase Chain Reaction
PCR
What is the goal of PCR?
Amplification of a region of target DNA
PCR
What are the steps for PCR?
1) Heat DNA to seperate strands
2) Add synthetic DNA oligonucleotide primers
3) Add Taq polymerase to catalyze 5’ -> 3’ DNA synthesis
PCR
What can you do after you have multiple copies of DNA made from PCR?
After you have multiple copies you can cut with restriction enzymes to insert into plasmid
Argose Gel Electrophoresis
What is the goal of Gel electrophoresis?
To determine the size of fragments
Argose Gel Electrophoresis
What are the steps of Gel electrophoresis?
1) Load DNA into wells at one end of argose gel
2) Connect to power supply
- Cathode (negative charge) = at the same end of the gel as the wells
- Anode (positive charge) = at the opposite end of the gel from the wells
3) DNA moves toward anode and movement speed/final positions depend on size
Argose Gel Electrophoresis
How is DNA normally charged?
Negatively
Argose Gel Electrophoresis
Are the longer molecules at the longer vs. shorter molecules in the Electrophoresis plate?
Longer = at the top
Shorter = at the bottom
Cloning the sequence of genes
DNA contains all introns and exons
How can you create recombinant DNA with just exons?
Complementary DNA (cDNA)
Cloning the sequence of genes
How is complementary DNA generated?
From mature mRNA
- no introns, only coding sequences
Cloning the sequence of genes
What does this process use?
Uses reverse transcriptase (make DNA from RNA)
- Can study expression
Recombinant protein purification
What might a plasmid utilize to control over-expression of proteins?
Some plasmids utlize an inducible promoter to control over-expression of proteins
Recombinant protein purification
What 3 techniques are used in Recombinant protein purification?
1) Epitope
2) Affinity chromatography
3) Green fluorescent proteins
Recombinant protein purification
What do Epitopes do?
Facilitate protein identification, visualization, and purification
- Tags recognized by specific antibodies
Recombinant protein purification
What does Affinity chromatography do?
Separation/purification
Recombinant protein purification
What does green florescent proteins do?
Visualize
Mutagenesis
What is mutagenisis?
To produce a mutation (deliberately and experimentally)
Mutagenesis
What occurs in oligonucleotide-directed mutagenisis?
- Insert mutagenic primers into plasmid
- Synthesize a new DNA strand with the mutagenic primer
Mutagenesis
What occurs in CRISPER mutagenisis?
1) Guide RNA = tells CAS where to cut, enabling precise targeting
2) CAS nuclease = cuts DNA
- A single strand cut allows recombined DNA to be introduced
- A double strand cut can lead to “non-homologous end joining”
DNA Sequencing
What are the steps of Sanger DNA sequencing?
1) Prepare a DNA sample and synthesize DNA with dNTPs
2) Add ddNTPs (tagged with florescent) to terminate synthesized DNA
3) Separate resulting DNA fragments by size and identify the florescent tags
DNA Sequencing
What are the steps of NGS sequencing?
DNA fragmented, florescent tagged synthesis is read, overlaps -> contigs, mapped onto reference
Gene Expression change
How do you measure changes in gene expression?
- Specifically how much mRNA is in expression
Quantitative Real-Time – Polymerase Chain Reaction utilizes florescent probes to monitor amplification
Gene Expression change
How do you measure changes in gene expression?
- Specifically compare expression across an entire transcriptome
Microarray analysis
Human genome
What are polymorphisms?
Small variations in gene sequence that lead to changes in phenotype and variation in populations
- Distinct
- Depends on reference
Human genome
What are Haplotypes?
Groupings of multiple SNPs
Think of blocks of the genome
- Generally defined as sets of genes inherited tg
Human genome
What are tag SNPs?
unique and define the haplotype
