8.3-4 Genome Project and Gene Technologies

Question 1

What is a genome map?

Answer 1

A complete map of all genetic material in an organism (called the genome)

Question 2

What are bioinformatics?

Answer 2

The science of collecting and analysing complex biological data such as genetic code

Question 3

What is whole shotgun sequencing?

Answer 3

Many copies of the genome cut up into small fragments to be sequenced
Fragments are assembled using computer algorithms to match overlapping sections

Question 4

What are SNPs

Answer 4

Single nucleotide polymorphisms
They are single base variations in the genome that are associated with disease and other disorders

Over 1.4 million in the human genome

Question 5

How can SNP be used in biology?

Answer 5

In medical screening SNPs allow quick identification of potential medical problems

Allows for early intervention for treatment

Question 6

What information can we apply from genomes?

Answer 6

Identification of potential antigens in order to produce vaccines

Sequence of amino acids in polypeptides can be predicted - allowing for synthetic biology

Question 7

Why are complex organisms more difficult to determine the genome and proteome?

Answer 7

We have many non-coding genes

Introns

Question 8

How does proteome differ in cells?

Answer 8

Did to selective gene expression not all genes are switched on in all cells therefore proteins aren’t always produced

Question 9

Why is it easier to sequence the genome of a bacteria?

Answer 9

The prokaryotes have just one circular piece of DNA

With no Introns

Question 10

How can the genome of an extremophile be useful?

Answer 10

If they can withstand extreme/toxic environments

They have a potential to clean pollutants or manufacture biofuels

Question 11

What is Encode?

Answer 11

Encyclopaedia of DNA elements

It is a catalogue of regions of the human genome that control the function if genes

Question 12

What can the human genome project help with?

Answer 12

Screening for mutated sequences and carriers

Screening for disorders before symptoms appear

Question 13

What are some ethical considerations of the human genome project?

Answer 13

People being discriminated against

Misuse and ownership of genetic material

Question 14

What is recombinant DNA technology?

Answer 14

It involves the transfer of fragments of DNA from one organism, or species, to another

Made possible due to DNA being universal

Question 15

What ways can fragments of DNA be produced?

Answer 15

Conversion of mRNA to complementary DNA (cDNA)

Using restriction enzymes

Using a ‘gene machine’

Question 16

How is mRNA converted to complementary DNA (cDNA)?

Answer 16

Isolate mRNA from cytoplasm using an ultracentrifuge

Using reverse transcriptase (found in HIV) mRNA acts as a template strand for cDNA to be formed

Using DNA polymerase double stranded DNA is formed from the single cDNA strand

Question 17

What is a key point about mRNA being converted to complementary DNA (cDNA)?

Answer 17

There are no introns produced in the gene

(We took the genetic information from an already spliced mRNA strand)

So the can be cloned into prokaryotic bacteria - also no Introns

Question 18

How are fragments of DNA produced from restriction endonucleases?

Answer 18

Restriction endonucleases are enzymes extracted from bacteria

They cut the DNA at specific sequences (enzymes are complementary) - and they cut to keep the entire gene intact
Best cuts = sticky ends (staggered like stairs)

The sticky ends allow two ends that are complementary to form a covalent bond and ‘rejoin’ (if the same restriction endonuclease was used)

Question 19

What is the gene machine?

Answer 19

The allow genes to be artificially manufactured

Amino acid sequence of the protein
|
mRNA codons
| 
Responding DNA sequence

Question 20

In the gene machine what does a bacterial plasmid act as?

Answer 20

A vector

It carries one gene to another

Question 21

What are gene markers? Types?

Answer 21

An extra gene in the plasmid that are easily identifiable

Antibiotic resistance genes
Fluorescence gene markers
Enzyme markers

Question 22

How do antibiotics resistance genes work as markers?

Answer 22

If you cut a plasmid with a restriction endonuclease to insert a gene
You could cut through an antibiotic resistance gene
= the gene is no longer functional and no longer resistant to that bacteria

So the plasmids that are killed by a bacteria are the recombinant plasmids we want

Question 23

Overview of replica plates in identification using antibiotic resistance?

Answer 23

The plasmids taken up by bacterial cells won’t all be recombinant
Culture the bacteria on an agar plate with some antibiotic in order to isolate the bacteria with plasmids taken up (they are resistant to it)
A replica plate is made and another antibiotic is added which should kill the recombinant plasmids
We have to then go back to the previous plate for the colonies killed on the second plate

Question 24

Information about fluorescent gene markers?

Answer 24

The gene originates in jellyfish

Default plasmid = fluorescent
Cut plasmid with gene = not fluorescent

Advantage - easy to identify

Question 25

Information about enzyme markers?

Answer 25

Enzyme in tact = colour change

Enzyme cut in a recombinant plasmid = no colour change

Question 26

How is a recombinant bacteria cultured after it is formed? Factors?

Answer 26

Fermenters

Supply of O2
Condenser to regulate heat
pH regulator

(Don’t want to denature enzymes)

Question 27

How do we ‘amplify’ DNA fragments after they are formed?

Answer 27

In vivo

In vitro

Question 28

After a recombinant plasmid is formed what happens?

Answer 28

The recombinant plasmid is reintroduced into a bacterium

Using electroporation
It facilitates the uptake of the plasmid by increasing permeability of bacterial membranes = more chance of success

Achieved via addition of calcium chloride and rapid temperature change from 0-40 degrees

Question 29

What is in vitro cloning?

Answer 29

Uses the Polymerase chain reaction (PCR)
In a thermocycler DNA fragments are separated into single strands by heating to 95 degrees - breaking hydrogen bonds
It is cooled to 55 degrees where DNA primers bind to the ends of the single strands
It is reheated to 72 degrees where DNA polymerase joins the nucleotides together forming a double stranded fragment using phosphodiester bonds and complementary base pairings - repeat around 30 times

Question 30

Why are DNA primers used in PCR (in vitro cloning)? How many are needed?

Answer 30

It gives the polymerase something to bind to in order to start replication there

2 per fragment are needed as there are two strands formed with different base sequences

Question 31

Why is 72 degrees used in the 3rd stage of PCR (in vitro cloning)?

Answer 31

It is the optimum temperature for TAQ polymerase to function at

TAQ polymerase is from a bacteria that live in hot springs

Question 32

Why is in vitro cloning often used?

Answer 32

When you have a very small amount of DNA at a crime scene and you need a lot more to analyse

Question 33

What is in vivo cloning?

Answer 33

Isolated DNA fragments are placed into plasmids by:
The plasmid and gene is cut using the same restriction endonuclease to create complementary sticky ends so the DNA and vector are complementary to join together
The fragments and plasmids are incubated together and if the fragment is taken up it is sealed using DNA Ligase forming phosphodiester bonds
A recombinant DNA molecule is created

Question 34

What are the advantages and disadvantages of in vivo cloning?

Answer 34

Very accurate
Can introduce genes into different organisms - expressed
No risk of contamination

Slow - relies on bacteria regeneration time
Monitoring of cell growth
Needs a large sample
Needs more purification

Question 35

What are the advantages and disadvantages of in vitro cloning?

Answer 35

Fast
Works on small quantities
Simple purification

Inaccurate
Risk of contamination

Question 36

What are ethical issues of using recombinant DNA technology?

Answer 36

GM crops could affect food chains and could lead to pesticide-resistant weeds
Unknown long term effects of altering an organisms genome

Question 37

What are two techniques that are important in identifying and locating particular alleles of genes in screening?

Answer 37

DNA probes

DNA hybridisation

Question 38

What are DNA probes?

Answer 38

Short pieces of DNA (fluorescently or radioactively labelled - using P32 on the phosphate group)
That binds to a specific base sequence of the ‘faulty DNA’
The X-ray film will then be exposed

Question 39

What can DNA probes be used for?

Answer 39

To detect heritable conditions of health risks

Question 40

What should we be aware of with genetic screening?

Answer 40

Genetic councelling

Question 41

Why can we do genetic fingerprinting?

Answer 41

Due to Variable number tandem repeats (VNTRs) - short repeating sequences
It is the 95% of DNA that doesn’t code for anything

This is UNIQUE to each individual

Question 42

How is genetic fingerprinting carried out generally?

Answer 42

Gel electrophoresis - separates the DNA fragments to their size using electric current

It has:
Sample wells: for the DNA
Electrodes
Buffered/isotonic solution 
Agar gel

Question 43

How is genetic fingerprinting carried out specifically?

Answer 43

DNA is extracted (maybe undergoes PCR) and restriction endonucleases cut the DNA into fragments
They are separated in gel electrophoresis
The fragments are transferred to a nylon membrane - known as Southern Blotting
DNA probes are added and excess DNA rinsed
The radioactive probes are placed on x-ray film and development reveals dark bands only where DNA is

Question 44

How and why does DNA move in gel electrophoresis?

Answer 44

DNA moves from the negative electrode to the positive electrode

This is due to DNA being negatively charged (because of the phosphate group)

Question 45

What is significant about the position of the fragments in genetic fingerprinting?

Answer 45

The larger fragments are at the top - not moved as far from the well
The smaller fragments are further away

Due to the heaviness of the fragments

Question 46

What are the main uses of DNA fingerprinting?

Answer 46

Forensics
Medical Diagnosis
Plant and animal breeding
Paternity tests