Manipulating genomes Module 6

Question 1

3 techniques to study genes?

Answer 1

The polymerase chain reaction (PCR)
Gel electrophoresis
Cutting out DNA fragments, using restriction enzymes

Question 2

How can multiple copies of a DNA fragment be made, using PCR?

Answer 2

A reaction mixture is set up, containing the DNA sample, free nucleotides, primers and DNA polymerase

DNA mixture is heated to 90 degrees, to break the hydrogen bonds between the 2 strands of DNA

Mixture is then cooled to between 50 and 65 degrees, so the primers can anneal (bind) to the strands

Reaction mixture heated to 72 degrees so DNA polymerase can work

DNA polymerase lines up free nucleotides with alongside each template strand, complementary base pairing means new complementary strands are formed

2 new copies of the fragment of DNA are formed, and one cycle of PCR is formed

Question 3

What are primers?

Answer 3

Short pieces of DNA that are complementary to the bases at the start of the fragment you want

Question 4

What’s DNA polymerase?

Answer 4

An enzyme that creates new DNA strands

Question 5

Why is it useful that DNA polymerase doesn’t denature at high temperatures?

Answer 5

Useful as cycles of PCR can be carried out without having to use new enzyme each time

Question 6

What happens when PCR is repeated?

Answer 6

Now have 4 strands (2 new and 2 original)

So next cycle there will be 8 then 16 then 32…

Question 7

What is electrophoresis?

Answer 7

Procedure that uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size

Question 8

Explain electrophoresis steps

Answer 8

Row of wells is created at the end of some agrose gel, which is in a gel box/tank, with the wells nearest to the negative electrode

Add loading dye to each DNA sample, helps the sample sink to bottom of wells making them easier to see. Add a set amount of each DNA fragment solution to each well

Pass an electrical current through the gel, DNA fragments are negatively charged so will move towards the positive electrode.
Small DNA fragments will move faster and travel further, through the gel causing them to separate by size, turn power off when 2cm from end. Stain the DNA fragments

The bands of different DNA fragments will now be visible

Question 9

How is electropheresis carried out on proteins?

Answer 9

Proteins can be positively or negatively charged, so before put in wells are mixed with a chemical that denatures the proteins so they all have the same charge

Used to identify blood or urine samples for disease

Question 10

How can restriction enzymes be used to cut out DNA fragements?

Answer 10

Some sections of DNA have palindromic sequences of nucleotides, these sequences consist of anti-parallel base pairs (base pairs that read the same in the opposite direction)

Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places

Different restriction enzymes cut at different specific recognition sequences because the shape of the recognition sequence is complementary to an enzyme’s active site.

If recognition sequences are present at either side of the DNA you want, you can use restriction enzymes to separate it from the rest of the DNA

DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment via a hydrolysis reaction

Sometimes cut leaves sticky ends (small tails of unpaired bases at each end of the fragment), as cut in a zig zag motion. Sticky ends can be used to anneal the DNA fragment to another piece of DNA, that has sticky ends with complementary sequences

Question 11

What’s a genome?

Answer 11

All the genetic material in an organism

Question 12

How can DNA profiles be created, and how do they distinguish between 2 different people?

Answer 12

Some of an organism’s genome contains repetitive non-coding base sequences

The number of times these repeat differ in person to person, therefore their length in nucleotides does aswell

The number of times a sequence is repeated (therefore the amount of nucleotides) at different loci (specific place), can be analysed using electrophoresis. Creating a DNA profile

Question 13

Why is the probability having the same DNA profile very low?

Answer 13

Because having the same number of sequence repeats at each locus is very low

Question 14

How can DNA profiling be used in forensic sciences?

Answer 14

DNA taken from all possible suspects

DNA is isolated

PCR is used to amplify multiple areas containing different sequence repeats

PCR products are run on electropheresis gel, and the profiles are compared to see if any match (have the same bands)

Question 15

How DNA profilling be used to identify to see if samples have come from the same species?

Answer 15

The more similar the bands, the more likely they are from the same species

Question 16

How can DNA profilling be used in medical diagnosis?

Answer 16

A DNA profile can refer to a unique pattern of several alleles

It can be used to analyse the risk of genetic disorders, where specific mutation is unknown or several mutations may have occurred, because it identifies a broader, altered genetic pattern

Question 17

What is genetic engineering?

Answer 17

The manipulation of an Organism’s DNA

Question 18

What’s a transformed organism?

Answer 18

Organisms that have had their DNA altered by genetic engineering, so have recombinant DNA (DNA formed by joining together DNA from different sources)

Question 19

Brief outline of how genetic engineering process?

Answer 19

Involves extracting a gene from one organism, and then inserting it into another organism, normally of a different species. Or genes can also be manufactured instead of extracted from another organism.

The organism with the inserted gene will now produce the protein coded for by that gene

Question 20

In depth description of how first step of genetic engineering works?

Answer 20

DNA fragment containing the desired genes is isolated using restriction enzymes

Question 21

In depth explanation how the second step of Genetic engineering works?

Answer 21

The DNA fragment is inserted into a vector DNA (Vector is something used to transfer DNA into a cell, eg. plasmids (small circular molecules of DNA in bacteria, or bacteriophages (viruses that infect bacteria))

The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment that containing the desired gene. So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene

The vector DNA and DNA fragment are mixed together with DNA ligase

DNA ligase joins up the sugar-phosphate backbone of the 2 bits, = ligation

Question 22

In depth explanation how the third step of genetic engineering works?

Answer 22

The vector with the recombinant DNA is then used to transfer the gene into the bacterial cells

If a plasmid vector is used, bacterial cells have to be convinced to take in the plasmid vector, and it’s DNA

Eg. suspension of bacterial cells is mixed with plasmid vector in a machine called an electroporator

The machine is switched on and an electrical field is created in the mixture, increasing the permeability of the bacterial cell membranes, and allows them to take in the plasmids, this is called electroporation

With a bacteriophage plasmid, the bacteriophage will infect the bacterium by injecting it’s DNA into it, the phage DNA then intergrates with the bacterial DNA

Question 23

How can genetic engineering be used to improve plant yield?

Answer 23

Inserted with a gene that produces a protein that is toxic to some insects

Also positive ethical issue, as less chemical pesticides that farmers have to use on their crops

Negative ethical issues, promotes monoculture (only one type of crop planted, decreasing biodiversity, and leaves all crops vunerable to one disease as all genetically indentical

Question 24

How can genetic engineering be used on animals?

Answer 24

They can be used to be make proteins, DNA fragments for protein injected into animal embryo, the embryo is then implanted into a female adult, when offspring is born, it’s tested to see if it can produce the protein. If it can it is reproduced using selective breeding

The protein is then extracted from the milk and put in the medicine

Positive ethical with pharming, is that drugs can be made in larger quantities, saving more people

Negative ethical issue, is that can harmful side effects o animals, and makes animals only assests

Question 25

Example of how genetic engineering be used be carried out on pathogens for research?

Answer 25

Tumour cells have receptors on their membranes for the polio virus, so poliovirus will recognise and attack them

So can genetically engineer the poliovirus, to deactivate the genes that cause poli,but it will still attack and kill cancer cells

Question 26

Ethical pros and cons of genetically altering pathogens?

Answer 26

Positives- new disease can now be cured

Negatives-
scientists doing research become infected and spread the disease
Genetically modified version of pathogen could revert to original form, and cause an outbreak of disease
Could be used in bio-warfare

Question 27

Ethical pros and cons of companies patterning genetically engineered organisms?

Answer 27

Positives- there’s more incentive to create genetically engineered organisms, as more money so more scientific breakthroughs are made

Negatives- In third world countries, may have to rely on genetically engineered organisms ie crops, but pattern regulations mean they have to pay excessive amounts

Question 28

What’s gene therapy?

Answer 28

Altering alleles inside cells, to cure genetic disorders

Question 29

How do you use gene therapy if the problem is caused by 2 recessive alleles?

Answer 29

Add a working dominant allele

Question 30

How do you use gene therapy if it’s caused by a dominant allele?

Answer 30

You can silence the dominant allele by adding a piece of DNA in the middle so it doesn’t function anymore

Question 31

How do you get an allele inside a cell?

Answer 31

Using a vector

Question 32

What’s somatic therapy?

Answer 32

Altering the alleles in body cells, particularity targeting the cells most affected by the disorder

Somatic therapy doesn’t affect the individuals sex cells, so any offspring could inherit the disease

Question 33

What’s germ line therapy?

Answer 33

Altering the alleles in the sex cells, this means every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t inherit the disease, currently illegal

Question 34

Positive ethical issues of gene therapy?

Answer 34

Prolong lives and increase quality of life of people with genetic disorder

Germ therapy means people can have children without spreading disease, therefore also reducing the amount of people with the disease

Question 35

Negative ethical issues of gene therapy?

Answer 35

Could be used not only for medical reasons, but cosmetic reasons

Very expensive, money could be used more wisely

In somatic therapy effects may be short lived, and have to undergo multiple treatments

The body could identify vectors as foreign bodies and start an immune response against them

Allele may be difficult to insert into specific body cell, or entered into wrong place, causing cancer

Inserted allele could be overexpressed, producucing too much of the missing protein

Question 36

How can DNA be sequenced using the chain-termination method?

Answer 36

The following mixture is added to 4 separate tubes:
A single stranded DNA template

Lots of DNA primers

DNA polymerase

Free nucleotides

Florescently labelled modified nucleotide, like a normal nucleotide, but once it’s added to a DNA strand no more bases can be added after that, (a different modified base is added to each tube A,T,C,G

The tubes undergo PCR which produces many strands of different length DNA, because each one terminates at a different point

The DNA fragments in each tube are separated by electropheresis, and visualised under UV light

The complementary base sequence can be read from the gel as smallest length is first and longest is last

(Have to flip to find sequence of original strand)

Question 37

How can gene sequencing techniques be used to find whole genomes?

Answer 37

A genome is cut into smaller fragments using restriction enzymes

The fragments are inserted into different bacterial artificial chromosomes (man made plasmids)

The BAC’S are then inserted into bacteria-each bacterium containing a BAC with a different DNA fragment

The bacteria divide, creating colonies of identical cells that all contain a specific DNA fragment, together the different colonies form genomic DNA libary

DNA is extracted from each each colony, and cut up using restriction enzymes producing overlapping pieces of DNA

Each piece of DNA is sequenced using chain-termination method, and put together back in order to give full sequence from BAC

Sequences from all BAC’s put together to form the whole genome

Question 38

Explain how faster whole genomes sequencing techniques have been developed to improve chain termination technique?

Answer 38

Chain termination technique, is now automated and faster, as each tube contains all the terminator sequences, and instead having a running gel, machine just reads them out

Question 39

Explain pyrosequencing the faster whole genome sequencing technique

Answer 39

Section of DNA is cut into fragments, split into single strands, and then a strand from each fragment is attached to a small bead

PCR is used to amplify the DNA fragments, and each bead, then each bead is put into a separate well

Free nucleotides are added to the wells and attach to the DNA via complementary base pairing

Wells also contain specific enzymes which cause light to be given off when a certain base attaches

Computers analyse light intesities and allows them to calculate the whole genome

Question 40

What is synthetic biology?

Answer 40

The construction of new biological systems using DNA made from scratch

Question 41

How does sequencing genomes aid synthetic biology?

Answer 41

Amino acids are coded for by triplets of bases on a gene

So from the sequence of a gene can predict the primary structure of a protein

Question 42

How can comparing sequenced genes or whole genomes be useful?

Answer 42

To study genotype/phenotype relationships
In epidermiological studies (study of health and disease within a population)
To help understand evolutionary relationships, more similar genomes = more closely related

Question 43

Explain pyrosequencing the faster whole genome sequencing technique?

Answer 43

Section of DNA is cut into fragments, split into single strands, and then a strand from each fragment is attached to a small bead

PCR is used to amplify the DNA fragments, and each bead, then each bead is put into a separate well

Free nucleotides are added to the wells and attach to the DNA via complementary base pairing

Wells also contain specific enzymes which cause light to be given off when a certain base attaches

Computers analyse light intesities and allows them to calculate the whole genome