Chapter 20: Recombinant DNA Technology

Question 1

These cut DNA in specific spots.

Answer 1

Restriction enzymes

Question 2

What are the uses of restriction enzymes?

Answer 2

1. Isolate large quantities of specific genes/other DNA sequences to isolate proteins
2. Study gene organization and function, factors regulating gene expression, and the nature and function of encoded proteins

Question 3

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’.

Answer 3

palindromes

Question 4

DNA molecules that accept DNA fragments and replicate inserted DNA fragments when placed into host cells are called _____.

Answer 4

vectors

Question 5

Which vector holds the smallest number of inserts?

Answer 5

Plasmids

Question 6

Which vector holds the largest number of inserts?

Answer 6

Artificial yeast chromosomes

Question 7

What are the four different types of vectors?

Answer 7

Plasmids, phages, cosmids, and artificial yeast chromosomes

Question 8

How are phages loaded with the DNA needed?

Answer 8

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.

Question 9

What advantage does a cDNA library have over a genomic library?

Answer 9

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.

Question 10

How does reverse transcriptase work in making a cDNA library?

Answer 10

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.

Question 11

This method of copying DNA allows us to quickly amplify a small region of the genome via amplification.

Answer 11

PCR

Question 12

What three things are necessary for PCR?

Answer 12

Information about the sequence to be cloned, use of that sequence to synthesize two oligonucleotide primers, and heat-stable DNA polymerase.

Question 13

Why do we need the oligonucleotide primers for PCR?

Answer 13

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.

Question 14

Why is the DNA placed in a machine that heats it?

Answer 14

That way, denaturation can be automated (helicase is not necessary). The heat will separate the strands.

Question 15

Why must our polymerase be heat resistant?

Answer 15

Otherwise, it too would denature in the heat.

Question 16

What is added after the DNA has been denatured into single strands in a PCR reaction?

Answer 16

The primers, followed by polymerase.

Question 17

What happens after the first cycle of PCR?

Answer 17

The process is repeated about 25 times, replicating the DNA we started with.

Question 18

What is PCR useful for?

Answer 18

Amplification to detect mutations and perform disease diagnostics, cancer therapy monitoring, detecting infections, and forensic science.

Question 19

This allows us to identify and quantify mRNAs.

Answer 19

cDNA cloning

Question 20

Poly-T primers are necessary in cDNA cloning for what?

Answer 20

Complimenting the poly-A tail of the mRNAs.

Question 21

In cDNA cloning, this digests the RNA strand, leaving just the desired DNA.

Answer 21

RNAaseH

Question 22

In this technique, mRNA is turned into DNA with reverse transcriptase and then a PCR is run on it.

Answer 22

Reverse transcription PCR (RT-PCR)

Question 23

What is a limitation of PCR?

Answer 23

We must know some information about the nucleotide sequence of the target DNA in order to make the primer.

Question 24

Why can’t PCR amplify long DNA segments?

Answer 24

Polymerase in a chain reaction can only extend primers for a short distance.

Question 25

How is electrophoresis performed?

Answer 25

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.

Question 26

This technique allows you to take any DNA you want and identify where in the DNA a specific sequence is located.

Answer 26

Southern blot

Question 27

List the steps of a Southern blot.

Answer 27

1. Cut samples with restriction enzyme
2. Perform electrophoresis on fragments
3. Place gel on a sponge and transfer to filter with buffer
4. Place the copy in a ziplock bag with the probe, wash off excess, and place on X-ray film.
5. Look at which bands of the gel hybridized with the radioactive probe.

Question 28

This technique allows you to detect insertion or deletion mutations without having to clone anything, due to the probe that is used.

Answer 28

Southern blot

Question 29

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.

Answer 29

Dideoxynucleotide chain-termination sequencing

Question 30

What is special about dideoxynucleotides?

Answer 30

They are missing the 3’ OH, preventing addition of another nucleotide.

Question 31

Dideoxynucleotide sequencing allows for around _____ nucleotides to be sequenced.

Answer 31

1000

Question 32

This technique allows you to look for a specific mutation or sequence by amplifying a short section of DNA using heat to denature it.

Answer 32

PCR

Question 33

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.

Answer 33

Southern blot