Ch 10: Analyzing the Structure and Function of Genes

Question 1

Which of the following statements about PCR is not true?
A) PCR can be used to clone a gene
B) PCR can be used to amplify a DNA sequence
C) PCA can use used to detect the presence of a virus in a blood sample
D) PCR can be used to sequence a genome

Answer 1

D: PCR generates many identical copies of relatively short DNA sequences. It is used to help amplify the strands that are being sequenced, but does not actually help us figure out the sequences

Question 2

In the polymerase chain reaction, what is used to separate the two strands of double-stranded DNA molecule

Answer 2

Heating

Question 3

What cannot be determined by comparing a nucleotide sequence of a chosen gene to sequences available in public databases?

Answer 3

The gene’s precise role in the physiology or development of the organism. But you can tell the likely function based on the gene’s known activity in other organisms, and other information as well.

Question 4

How does automated dideoxy sequencing differ from the original method?

Answer 4

It uses a mixture of chain-terminating nucleotides, each with its own label

Question 5

What technique allows a researcher to measure the presence of specific mRNAs in single cells in a tissue?

Answer 5

in situ hybridization: this allows researchers to observe the presence of specific mRNAs in particular cells in a fixed tissue preparation. It uses labelled probes to detect complementary nucleotide sequences within a cell or tissue, even on an isolated chromosome. It can show the precise location of genes on chromosomes and of RNAs in tissues and cells

Question 6

Why is genetic engineering in plants simpler than it is in animals?

Answer 6

Transgenic plant cells can be grown from almost any type of plant cell transfected with DNA in culture.

Question 7

Brief overview of what DNA cloning allows us to do

Answer 7

A DNA fragment can be cut into specific sequences, and can allow any sequence to be selected from millions of other sequences and produced in unlimited amounts in pure form.

These fragments can be joined in vitro using DNA ligase, and they can be maintained and amplified by inserting them into a larger DNA molecule, such as a plasmid, that is capable of replication. This is usually introduced into a bacterium, so the DNA is replicated at each cell division.

Question 8

What is a genomic library?

Answer 8

A collection of cloned fragments of chromosomal DNA representing the complete genome of an organism. It is often maintained as millions of clones of bacteria, each carrying a different fragment of the organism’s genome.

Question 9

What are cDNA libraries and what is the difference between those and DNA libraries?

Answer 9

cDNA libraries contain cloend DNA copies of hte total mRNA of a particular cell type or tissue. These are different from genomic libraries because they contain mostly the protein-coding sequences, and lack the regulatory DNA sequences and promoters. They are useful when the cloned gene is needed to make a protein.

Question 10

What is PCR amplification?

Answer 10

polymerase chain reaction. It is a form of DNA amplification that is carried out ina test tube using a purified DNA polymerase. Synthetic primers bracket the DNA being replicated, and copies of this sequence the serves as templates for successive rounds of replication.

Question 11

What are transgenic organisms?

Answer 11

When cloned genes are altered in vitro, and stably inserted into the genome of a cell of organism to study their function.

Question 12

Describe the CRISPR system

Answer 12

Genes can be modified or deleted with high specificity by using guide mRNAs to promote the cut of a DNA molecule at a specific nucleotide sequence in the genome. The sequences can be modified or deleted

Question 13

cDNA

Answer 13

complementary DNA: DNA synthesized from an mRNA molecule, which means it lacks the regulatory sequences/introns that exist in genomic DNA

Question 14

Overview of Sanger (dideoxy) sequencing

Answer 14

uses DNA polymerase and a set of chain-terminating nucleotides

Question 15

DNA ligase

Answer 15

enzyme that seals nicks that arise in the phosphodiester backbone of a DNA molecue. In the laboratory, can be used to join together two DNA fragments.

Question 16

gene knockout

Answer 16

a genetically engineered animal in which a specific gene has been inactivated

Question 17

plasmid

Answer 17

small, circular DNA molecule that replicated independently of the genome. Used extensively as a vector for DNA cloning

Question 18

recombinant DNA

Answer 18

a DNA molecule that is composed of DNA sequences from different sources

Question 19

RNA interference (RNAi)

Answer 19

cellular mechanism activated by double-strand RNA molecues that results in the destruction of RNAs containing a similar nucleotide sequence. It is widely exploited as an experimental tool for preventing the expression of selected genes (gene slicing)

Question 20

RNA-Seq

Answer 20

sequencing technique used to determine directly the nucleotide sequence of a collection of RNAs

Question 21

transformation

Answer 21

process by which cells take up DNA molecules from their surroundings in a way that allows them to permanently express genes present on that DNA

Question 22

Why is DNA sequencing important?

Answer 22

Sequencing is one of the most influential technologies developed. You can sequence, DNA, RNA, learn the order of amino acids, and it helps us understand life

Question 23

Why do we want to know the sequence of DNA?

Answer 23

Gives us a basic understanding of life and tells us a lot a bout proteins: what kinds of proteins, where they are, what are the consequences on the cells.
It can help prevent disease, fix disease, maybe treat the patient.
It acts as a diagnostic.
Helps research and develop treatment.

Question 24

What aspect of DNA is the most important in terms of letting us sequence DNA?

Answer 24

The copying mechanism of DNA

Question 25

What is one of the major developments in sequencing DNA

Answer 25

Automation (and to some extent miniaturization). Understanding the hardware, the biology, and the hardware miniaturization are all important for developing further technologies

Question 26

Describe the Sanger method/ Chain Termination method

Answer 26

This whole process can happen in a test-tube, as long as everything that is needed for DNA replication is provided.
Key is use of dideoxynucleotide-triphosphates (ddNTPs)
ddNTPs have two hydrogen groups…they are missing the hydroxyl group that DNA has on the 3’ end and has a hydrogen instead.
- It begins with a DNA primer that is based on previously known sequence that the replication will continue. One challenge is to supply the DNA primer – we have to be able to design right primer (10-20 base pairs). The primer is a reverse complement of the start of what we want to sequence
- The DNA to be sequenced is cut into random-sized fragments.
- DNA polymerase is used to begin the replication process, and there is a mixture of normal DNA nucleotides with a small amount of ddNTPs mixed in.
- When the DNA polymerase adds one of the ddNTPs onto the end of the strand (this happens randomly as the next nucleotide that is picked up happens to be a ddNTP), the replication of that strand terminates, because nothing is able to be added onto the 3’ Hydrogen group.
- The result is a series of DNA fragments, all starting at the same point, and all ending at a random terminus some number of base pairs later when they pick up a ddNTP. The original process of this, done by hand, has four separate reactions that happen, each with the ddNTPs representing one of the base pairs.
- A sample from each of those reactions is put into a well at the end of a gel. Because DNA has a negatively charged phosphate backbone, when a current is run through the gel, the DNA fragments will begin to migrate to the other end of the gel. The smaller fragments are able to move through the gel more quickly, and so the fragments of DNA will separate themselves out by length into different bands.
- Enough reactions will make all the possible lengths, so comparing the relative lengths of each fragment and knowing the termination ddNTP lets you figure out the sequence

Question 27

How do ddNTPs cause chain termination?

Answer 27

DNA has 1 -OH group at the 3’, ddNTP just has an -H, so it’s less reactive.

Question 28

What is fluorescence?

Answer 28

Shine a higher energy light (lower wavelength) on the molecules which excites the electrons. The molecule then emits a photon at a longer wavelength
Different molecules take different wavelengths in and emit different wavelengths, which results in different colors.

Question 29

What is the significance of fluorescent sequencing?

Answer 29

It only uses one reaction, and a laser to detect the sequence, which starts to allow for automation

Question 30

If this is the chromatogram, what is the sequence of the DNA?

Answer 30

5‘-TCAGCACGTTA-3’

Question 31

What are some considerations with the quality of the chain termination method?

Answer 31

• The DNA primer must be specific to the beginning of the sequence that is desired, otherwise different segments will be sequenced together, confusing the results.
• DNA secondary structures can affect how well they is sequenced: when DNA is split into single strands, it can fold back into itself and impede replication.
• The labelled ddNTPs bases have to have a similar reaction efficiency or else the trace chromatogram read-out will result in unequal peaks and difficult shapes to interpret.

Question 32

What is the major challenge with the chain termination method of sequencing the genome?

Answer 32

as the fragments get longer, it is harder to distinguish effectively between the bands. This is because the relative difference in length between 800 nucleotides and 801 is much smaller than the relative difference between 4 nucleotides and 5. This means that there is a limit to the length of a fragment that can be sequenced at once: usually only about 1000 bases. This limits the speed of sequencing, and makes automation much more important. The human genome is about 3 billion bases long.

Question 33

What are some strategies for dealing with the issue of the length of the human genome when sequencing it?

Answer 33

1) Break the genome into smaller fragments (shotgun method)
2) Sequence those smaller pieces
3) Assemble the short fragments into a comprehensive chromosome, and a whole genome by looking at overlapping segments and deducing the order they go in.
4) Annotation: computational and experimental. The most difficult part is piecing together the different fragments.
5) The more that is sequenced, the easier the blanks are to fill in.

Question 34

What is the importance of DNA cloning?

Answer 34

Cloning the DNA allows for in vivo amplification: creating many copies of the DNA fragments from a small amount of source material.
Constructing a genomic library (representing an entire genome) is done by cutting up the DNA into smaller fragments, inserting the fragments into plasmids (a small circular DNA fragments) using ligase, and inserting those plasmids into bacteria where they get replicated many times as the bacteria cells divide.

Question 35

Describe Illumina sequencing

Answer 35

• Uses platform amplification: This is used to make many copies of DNA, in a way that is faster and cheaper than cloning in bacteria.
  o DNA samples are fragmented and prepared by adding ligate adaptors to both ends of the fragments
  o Then the DNA is attached to a surface where the fragments are randomly distributed
  o Unlabelled nucleotides and enzymes initiate bridge amplification
  o The fragments, attached by the adapter terminus, replicate, producing a double strand of the DNA fragment.
  o The double strand denatures into two single-strands, and the DNA replication process is repeated over and over, until there are dense clumps of identical DNA fragments, each nanometers in size
• Then the fragments are able to be sequenced:
  o Nucleotides are tagged with a fluorescent dye, and the hydroxyl group (the site where the next nucleotide is added) is blocked in a way that can be reversed later with another chemical reaction.
  o The next nucleotide in the sequence can attach to the DNA fragments, and, because the hydroxyl group is blocked (it is a terminator), it does not allow further nucleotides to attach. Each clump then glows a particular color, depending on which nucleotide was added..
  o Then the hydroxyl block is removed, which allows the next fluorescent tagged terminator to be attached, and the process is repeated, reading out one nucleotide at a time, on many different fragment sites.
  o This process allows hundreds of thousands of sequences to be read at the same time.

Question 36

How is RNA sequenced?

Answer 36

RNA can’t replicate the same way as DNA so we use reverse transcriptase to turn it back into DNA, and then can run DNA sequencing reactions

Question 37

What are some other applications for sequencing DNA and RNA?

Answer 37

Discovering what kind of chromatin are attached to what sequences of DNA
Transcriptomics (what proteins the cells are producing)

Question 38

How do we discover what types of chromatin are attached to what sequences of DNA?

Answer 38

• Different DNA sequences have different histone groups wrapped around them
• Cross-link the DNA to the histones (glue it), and digest everything else, leaving only the strand attached to the histones.
• Use antibodies to bind to histones of interest, and remove everything else
• Uncross-link the DNA, which frees the DNA that was attached to the histones of interest and allows those fragments to be sequenced, letting us know what DNA is attached to what histones.

Question 39

Transcriptomics

Answer 39

• Cells do not express every protein at every stage of life
• Knowing what they are expressing lets you know what they are doing at each stage
• Sequencing the RNA produced by the cells correlates to the DNA that is being expressed

Question 40

What is DNA secondary structure and why do you think it can affect sequencing?

Answer 40

When we heat DNA to split it into single-stranded DNA, it will sometimes fold back and bond to itself to form bends, etc. This can make primer have a hard time reading it.

Question 41

You’ve heard a lot about DNA sequencing (both in this class and probably in the news). DNA sequencing technology is very advanced and popular. However, you don’t hear too much about protein sequencing. Why is that? What is the fundamental differences between nucleic acids and proteins that allow DNA sequencing to be much more efficient than protein sequencing?

Answer 41

There is no copying mechanism on protein, no enzyme to read the sequence and make more of itself. This means you can’t amplify it, so it is inefficient to find the sequences of lots of them.

Question 42

Brief overview of the Sanger Sequencing method, and what is the difference between Deoynucleic acid and Dideoxynucleic acid

Answer 42

Deoxynucleotide has one hydroxyl group on its 3’ carbon, whereas dideoxynucleotide has hydrogen on its 3’ carbon, which does not allow addition of new nucleotide onto the 3’ end of the nucleic acid, and therefore. In the Sanger sequencing reaction, all four di-deoxy nucleotides are included in the reaction. Each one is labeled with a different fluorescent color so that one can identify immediately which base has been incorporated in a fragment of a certain size. The key to decoding a DNA sequence relies on two pieces of information: the color of the fluorescence indicates the base; the size of the DNA strand indicates the location of the particular base.

Question 43

What is the shotgun approach in genome sequencing and how does it work?

Answer 43

Shotgun sequencing is a method for sequencing long DNA strands developed by Craig Venter in 1970s. Since the chain termination method can only sequence a few hundred nucleotides, larger DNA was first fragmented, and then inserted into a convenient vector. Once the fragments were sequenced, the sequences will be deduced based on the overlapping ends of the fragments to obtain the continuous original long DNA sequence.

Question 44

What is next generation sequencing? How is it different from first generation sequencing

Answer 44

Next generation sequencing reads DNA sequence in situ as the nucleic acid extends, therefore, it knows the relative position of the bases. However, Sanger sequencing can only tell the position of a base by aligning fragments of different length together. Next generation sequencing allows efficient large scale sequencing.

Question 45

What are some applications of DNA sequencing?

Answer 45

Tracking diseases, uncovering evolutionary secrets, conserving wildlife.