CGE: Genome Projects and Making DNA Fragment

Question 1

Genome

Answer 1

Entire set of DNA, including all the genes in an organism.

Question 2

What was the Human Genome Project?

Answer 2

Mapped the entire sequence of the human genome for the first time.

Completed in 2003.

Question 3

Improvements in technology have allowed humans to do what?

Answer 3

Sequence the genomes of a variety of organisms.

Question 4

Gene sequencing methods only work on what?

Answer 4

Fragments of DNA - so to sequence the genome of an entire organism, it must be cut into smaller fragments first.

Question 5

Proteome

Answer 5

All the proteins made by an organism.

Question 6

Why is it relatively easy to determine the proteome of simple organisms, like bacteria?

Answer 6

They don’t have much non-coding DNA.

Question 7

Why is it useful that the proteome of simple organisms is relatively easy to determine?

Give an example.

Answer 7

Useful for medical research and development.

Eg. N.meningitidis group B bacteria cause meningitis B. Sequencing the genome of these bacteria helped researchers identify antigens for use in developing a vaccine against the disease.

Question 8

Why is it harder to translate the genome of complex organisms?

Answer 8

They contain large sections of non-coding DNA.

Also contain regulatory genes - determine when genes are turned on and off.

Therefore, it is harder to translate genome into their proteome because it is difficult to differentiate the coding genes from the non-coding and regulatory DNA.

Question 9

Briefly describe the differences between old sequencing methods and more modern methods:

Answer 9

Older = labour-intensive, expensive and only done on a small scale.

Newer = automated, more cost-effective and done on a larger scale.

Question 10

Pyrosequencing

Answer 10

A recently developed sequencing technique that can sequence around 400 million bases in a ten hour period.

Question 11

What does recombinant DNA technology involve?

Answer 11

Transferring a fragment of DNA from one organism to another.

Question 12

Why can the transferred DNA in recombinant DNA technology be used to produce a protein in the cells of the recipient organism?

Answer 12

Because the genetic code is universal and because transcription and translation mechanisms are similar too.

Question 13

Because genetic code is universal and translation and transcription are similar across organisms, what does this mean for recombinant DNA technology?

Answer 13

The transferred DNA can be used to produce a protein in the cells of the recipient organism.

Question 14

Transgenic organisms

Answer 14

Organisms that contain transferred DNA.

Question 15

What are the three ways that DNA fragments can be produced?

Answer 15

1. Using reverse transcriptase
2. Using restriction endonuclease enzymes
3. Using a gene machine

Question 16

Why is it often difficult to obtain DNA fragments containing the target gene?

What is easier to obtain?

Answer 16

Because most cells only contain two copies of each gene.

mRNA is easier to obtain, as cells contain may mRNA molecules which are complementary to the gene.

Question 17

Reverse transcriptase?

Answer 17

An enzyme that makes DNA from an RNA template.

Question 18

The DNA produced by reverse transcriptase is called what?

Answer 18

Complementary DNA (cDNA)

Question 19

How can DNA fragments be obtained using reverse transcriptase?

Answer 19

1. mRNA is isolated from cells.
2. mRNA is mixed with free DNA nucleotides and reverse transcriptase.
3. Reverse transcriptase uses the mRNA as a template to synthesis a new strands of cDNA.

Question 20

What does it mean if DNA has palindromic sequences of nucleotides?

Answer 20

It contains sequences that consist of antiparallel base pairs - these read the same in opposite directions.

Question 21

Restriction endonucleases

Answer 21

Enzymes that recognise specific palindromic sequences (recognition sequences) and cut (digest) the DNA at these places.

Question 22

Why do different restriction endonucleases cut at different specific recognition sequences?

Answer 22

Because the shape of the recognition sequence is complementary to the enzyme’s active site.

Question 23

How could you use restriction endonucleases to cut out a specific DNA fragment?

Answer 23

If recognition sequences are present at either side of the DNA fragment, restriction endonucleases can separate it from the rest of the DNA.

Question 24

How can DNA fragments be obtained using restriction endonucleases?

Answer 24

1. DNA sample is incubated with the specific restriction endonuclease.
2. Cuts the DNA fragment via a hydrolysis reaction.
3. Can leave sticky ends which can be used to anneal DNA to another piece of DNA.

Question 25

Anneal

Answer 25

Bind

Question 26

Sticky ends

Answer 26

Small tails of unpaired bases at the end of a DNA fragment.

Question 27

How can DNA fragments be obtained using a gene machine?

Answer 27

1. Required sequence is designed (if one does not already exist).
2. First nucleotide is fixed to a support - eg. a bead.
3. Nucleotides are added step by step in correct order - protecting groups are also added.
4. Oligonucleotides are produced.
5. Once complete, these are broken off from support and protection groups are removed.
6. Oligonucleotides can be joined to make longer DNA fragments.

Question 28

Protecting groups

Answer 28

Used in gene machines to make sure the nucleotides are joined at the right points, preventing unwanted branching.

Question 29

Oligonucleotides

Answer 29

Short sections of DNA that are roughly 20 nucleotides long.

Question 30

Gene machine

Answer 30

Database containing the necessary information to produce a DNA fragment - this does not have to be a naturally occuring DNA sequence.