Ch. 7 Studying Genes Flashcards
What is a genome?
A genome is ALL the genetic information of a cell, which codes for all functions. (46 chromosomes, mito DNA, plasmids in bacteria…)
What are the biotechnologies and how are they related to studying genes?
The techniques used to study genes are innumerable, but this section focuses on those related to biotechnologies: medicinal, agricultural, manufacturing use, etc.
What is gene cloning?
The replication of a gene from one species into another species.
What is a gene of interest?
A gene that is targeted and acquired, then inserted into a cloning vector using DNA ligase.
What does the insertion of a GOI into a cloning vector produce?
This produces recombinant DNA: DNA from two different sources that are combined into one piece.
How is a GOI actually cloned?
The recombinant vector (GOI and cloning vector) are placed into cells, and cells that have acquired the vector are selected for.
What are the three types of cloning vectors?
Plasmids, bacterial artificial chromosomes, and yeast artificial chromosomes.
When are plasmids used as a cloning vector?
Plasmids can be used for cloning up to ~10,000 bp.
What are bacterial artificial chromosomes and when are they used as a cloning vector?
They are specialized plasmids that can receive up to ~300,000 bp.
What are yeast artificial chromosomes and when are they used as a cloning vector?
They are plasmids modified to replicate in eukaryotes and can carry up to ~2,000,000 bp.
What are DNA libraries?
Collections of multiple large fragments of DNA that have been cloned.
How are DNA libraries created?
- whole genomes are cleaved into thousands of pieces
- each piece is cloned
Are DNA libraries required in today’s research?
No. Newer sequencing technologies DON’T require libraries, but early genome sequencing did require them.
What is cDNA?
complementary DNA; DNA complementary to cellular RNA
How can a cDNA library be made (4)?
It is made based on the mRNA present in a cell.
-mRNA is collected from cells (treat with DNase)
- reverse transcriptase is used to make a DNA copy of the transcripts
- mRNA is degraded and PCR is used to make a compliment to the cDNA
- the ds fragments are cloned into vectors
How can a primer for cDNA PCR be made?
Eukaryotic RNA has a poly-A tail, so and oligo-dT primer can be added to the sample.
What is PCR?
Polymerase chain reaction allows for the SPECIFIC amplification of desired portions of a genome.
What does conventional PCR require?
It requires DNApol, DNA primers that DNApol extends, and a target DNA sequence.
What is the “bare bones” process of PCR?
- the primers anneal to complementary sites in the DNA and determine what portion of the DNA is amplified
-template DNA is amplified by DNApol in vitro (in a tube)
Why is PCR used in biotechnology, research, diagnostics, and more?
It is used because of its high sensitivity!
What is RT-PCR?
Reverse transcription PCR; it uses an RNA template to make DNA. This is how a cDNA library is made.
What is quantitative PCR?
Also referred to as qPCR or real-time PCR; it records the amount of DNA present after each cycle to quantify the amount of DNA present in the original sample.
Does conventional PCR give you an amount of DNA?
No. It only tells you presence or absence of DNA.
If you combine qPCR and RT-PCR what can you determine?
You can determine the amount of a specific transcript (RNA) present in a sample.