Chapter 20: Recombinant DNA Technology Flashcards
These cut DNA in specific spots.
Restriction enzymes
What are the uses of restriction enzymes?
- Isolate large quantities of specific genes/other DNA sequences to isolate proteins
- Study gene organization and function, factors regulating gene expression, and the nature and function of encoded proteins
A form of symmetry that many recognition sequences form, resulting in the nucleotide sequence reads the same on both strands when read 5’ to 3’.
palindromes
DNA molecules that accept DNA fragments and replicate inserted DNA fragments when placed into host cells are called _____.
vectors
Which vector holds the smallest number of inserts?
Plasmids
Which vector holds the largest number of inserts?
Artificial yeast chromosomes
What are the four different types of vectors?
Plasmids, phages, cosmids, and artificial yeast chromosomes
How are phages loaded with the DNA needed?
Using a restriction enzyme, pretty much everything but replication information is removed from the phage’s DNA. It is replaced with human DNA and packed into the phage, and the phages are placed on a plate with a bacteria lawn. Clear spots show up, showing where the phage landed and killed the bacteria there.
What advantage does a cDNA library have over a genomic library?
Because cDNA is complementary to the nucleotide sequence of the mRNA being made by the cell, it represents the genes being expressed in the cell when the library was made. So instead of having the whole genome, it has only genes that were being expressed.
How does reverse transcriptase work in making a cDNA library?
Reverse transcriptase starts at the poly-A tail of an mRNA and begins synthesizing a complementary strand of cDNA. The mRNA is then digested with RNaseH, and polymerase I synthesizes a new strand to go with the cDNA, resulting in double stranded cDNA.
This method of copying DNA allows us to quickly amplify a small region of the genome via amplification.
PCR
What three things are necessary for PCR?
Information about the sequence to be cloned, use of that sequence to synthesize two oligonucleotide primers, and heat-stable DNA polymerase.
Why do we need the oligonucleotide primers for PCR?
They will base pair with only one region of the genome, and since polymerase needs the primer, it can only replicate in that one spot, isolating the section we need.
Why is the DNA placed in a machine that heats it?
That way, denaturation can be automated (helicase is not necessary). The heat will separate the strands.
Why must our polymerase be heat resistant?
Otherwise, it too would denature in the heat.
What is added after the DNA has been denatured into single strands in a PCR reaction?
The primers, followed by polymerase.
What happens after the first cycle of PCR?
The process is repeated about 25 times, replicating the DNA we started with.
What is PCR useful for?
Amplification to detect mutations and perform disease diagnostics, cancer therapy monitoring, detecting infections, and forensic science.
This allows us to identify and quantify mRNAs.
cDNA cloning
Poly-T primers are necessary in cDNA cloning for what?
Complimenting the poly-A tail of the mRNAs.
In cDNA cloning, this digests the RNA strand, leaving just the desired DNA.
RNAaseH
In this technique, mRNA is turned into DNA with reverse transcriptase and then a PCR is run on it.
Reverse transcription PCR (RT-PCR)
What is a limitation of PCR?
We must know some information about the nucleotide sequence of the target DNA in order to make the primer.
Why can’t PCR amplify long DNA segments?
Polymerase in a chain reaction can only extend primers for a short distance.
How is electrophoresis performed?
DNA is placed on a gel with two oppositely charged ends. Since the DNA is negatively charged, it will move towards the positive end. Larger fragments of DNA move slower than smaller ones. This can be used to determine the size of a piece of DNA.
This technique allows you to take any DNA you want and identify where in the DNA a specific sequence is located.
Southern blot
List the steps of a Southern blot.
- Cut samples with restriction enzyme
- Perform electrophoresis on fragments
- Place gel on a sponge and transfer to filter with buffer
- Place the copy in a ziplock bag with the probe, wash off excess, and place on X-ray film.
- Look at which bands of the gel hybridized with the radioactive probe.
This technique allows you to detect insertion or deletion mutations without having to clone anything, due to the probe that is used.
Southern blot
This is the technique where artificial dideoxynucleotides are put in instead of the normal one in a few strands, stopping synthesis and allowing you to read off the colors.
Dideoxynucleotide chain-termination sequencing
What is special about dideoxynucleotides?
They are missing the 3’ OH, preventing addition of another nucleotide.
Dideoxynucleotide sequencing allows for around _____ nucleotides to be sequenced.
1000
This technique allows you to look for a specific mutation or sequence by amplifying a short section of DNA using heat to denature it.
PCR
This technique allows you to take any DNA you want and determine where in it a specific sequence is located by looking at the gel under ultraviolet light after tagging it.
Southern blot
