Manipulating genomes

Who was dna sequencing Discovered by ?

Fred Sanger

What is dna sequencing ?

to identify nucleotide sequences within a larger piece of DNA

What did Sanger use ?

4 seperate dishes:

• dna polymerase

• single stranded DNA

• Primer

• Modified DNA nucleotides

What is the difference between normal and modified nucleotides ?

Normal have a hydroxyl group on the third carbon which acts as a hook for the next nucleotide to join onto, the modified don’t have this so chain is terminated

What is a primer ?

Short single stranded length of DNA that binds to template DNA as a starter in a large piece of DNA.

Describe the shortest length DNA sequence creation

1. The primer binds to the single stranded DNA-template at the 5” end

2. Starting from the primer, DNA polymerase joins nucleotides that have paired up by complementary base pairing by forming phosphodiester bonds

3. The first nucleotide that was added was modified so the chain ends there

describe the process with a normal nucleotide of DNA sequencing (chain termination)

1. Same as before

2. Same as before

3. The chain continues until the modified nucleotide is added to the sequence

What is electrophoresis ?

Separating DNA fragments using electric current

Describe the process of electrophoresis

Add DNA fragments to the well at the cathode end. The shorter the fragment = the further they go as are faster.

At the anode there is a laser to illuminate the dyes to identify the specific modified nucleotide and it’s attached dye

What is pyrosequencing an example of ?

A high throughput method

What ingredients are needed to pyrosequencing

Same 4 but activated nucleotides which have two extra phosphoryll groups that are released upon the nucleotide binding to sequence

Describe pyrosequencing process (enzyme)

ATP sulfurylase converts pyrophosphate into ATP in the presence of APS

then luciferase converts luciferin to oxyluciferin in the presence of ATP

then apyrase digests any un used activated nucleotides and degrades them

Describe pyrosequencing method

1. Mechanically cut DNA length into fragments

2. Degrade fragments to be ssDNA

3. Add primer, incubate ssdna with correct enzymes substrates and activated nucleotides

4. If activated nucleotide is included the extra phosphoryll groups are released upon its addition

5. Apyrase

What is the use of comparing genomes ?

can work out evolutionary relationships by amplifying and sequencing and find out what diseases an individual may be susceptible to, which medicines work best and what side effects they may have

Application of gene sequencing

Human genome project

Work out evolutionary relationships

Synthetic biology: making useful biological devices and systems

comparisons between species

Predict amino acid sequence : read the codons to work out the amino acids in the sequence

What is genome sequencing purpose ?

Where we can determine the DNA sequence of a genome plus : mitochondrial,chloroplast and chromosomal DNA.

It allows us to to see similarities between genes in species to see what genes are conserved and to establish evolutionary relationships between species by comparison of their genomes

What do small changes in some genes lead to ?

A large change in function, eg FOXP2 is a transcription factor that controls hundreds of genes and is essential for development of language and speed in humans but is also present in mice and chimps

What are differences in organisms caused by ?

caused by shared genes working in different ways eg the FOXP2

Changes to regulatory regions of DNA leading to increased DNA expression

What variation do humans have ?

0.1%

What is an SNP ?

Single nucleotide polymorphism which is the location in DNA where substitution occurs which can alter protein, alter gene expression and can have no effect

What are epigenetics

The study into how behaviour and environment can cause changes that affect gene function.

Looks at changes that result in the gene being switched on and off

Name 3 types of epigenetics

1. Methylation of certain chemical groups in DNA prevents transcription factors from binding

2. Histone modification where DNA is tightly wrapped around histone proteins and is unreadable so switched off but if unwrapped, it is switched on. Adding chemical groups to histones determines whether gene is wrapped or unwrapped

3. Non coding RNA is attached to coding RNA and with other proteins, it breaks it down. The non coding RNA is important for gene expression therefore

How do you predict an amino acid sequence ?

The genome reveals the nucleotide sequence and we compare the triplets to our known amino acid code to identify them and reveal primary structure. To do this you have to know which parts of the sequence are introns (non coding) and which are exons(coding)

What is synthetic biology ? + examples

Designing and building useful devices and systems, you redesign organisms to make a substance or have a new useful function for us

1. Yeast can produce rose oil as a more sustainable way for the perfume industry

2. We can alter food to solve dietary problems like golden rice

What is information storage ?

The idea that digital info can be coded on a single strand of synthetic DNA

Each letter has a 8 digit binary code which we translate into a block of 5 Letters (ATGC)

1g of dna = 1 million CDs worth of info

What are some other uses of synthetic biology ?

Helping to create medicines such as E. coli and yeast making anti malarial drug artemisinin which had to previously be extracted from plant at certain life cycle stage

Creating novel proteins such as one similar to haemoglobin but without the bind of carbon monoxide

What is a downside of synthetic biology ?

Has risks of ethics and bio security so we need a board of advisors and regulations in place

What is PCR

An artificial replication method where small amounts of DNA get amplified and increase exponentially doubling each time

What does PCR require ?

Primer

DNA polymerase

Taq polymerase ?

DNA nucleotides

DNA sample

State the steps of PCR

1. Initially add sample to all the contents plus magnesium ions

2. Heat to 94-96C to break hydrogen bonds in sample, making two separate DNA strands however this is bad for DNA polymerase as it needs 35C !!

3. So cool it to 68 degrees so primer can anneal at the 5 prime end

4. We use taq polymerase instead from thermophilus aquaticus as it is thermophilic and can work in hotter conditions

5. We increase the temperature to 72C to suit the Taq polymerase and it forms phosphodiester bonds, creating a new complementary strand

6. This is a cyclic process so repeats

Name applications of PCR

• tissue typing- can match recipient and donor tissues before a transplant to reduce risk of rejection

• For forensic science

• For research: take extinct animal DNA eg from a bone and use to assess evolutionary links and relationships

• To identify viral infections: such as Covid as small amounts of viral DNA can be detected

• Epidemiology- to study the spread and variants of pathogens

• Oncology- to study genes that are linked to cancers

• Mutations- to use in a pre-implantation genetic screening for IVF to check for certain genetic conditions

What does electrophoresis require

Agarose gel

Buffer solution

Tank with oppositely charged electrodes

Power supply

Why is DNA attracted to the anode

Due to the negative charge from all the phosphates in the structure

Describe the method of electrophoresis

1. restriction enzymes cut DNA at recognition sites at 35-40C for one hour

2. Make agarose gel and pour into the tank with a comb at the cathode end to make wells + set

3. Pour buffer solution over the gel and remove comb

4. Add loading dye to digested DNA

5. Use a pipette to add DNA to wells (hovering above)

6. Connect electrodes to the power supply and leave to run for 6-8h on low or 2 on high voltage

7. Disconnect power and pour buffer away. Add dye to gel

What is SDS

A charged detergent that nullifies any surface charges on proteins to they can be separated based on molecular mass. It forms them all into a rod like shape so different shapes don’t affect the separation.

• used to diagnose sickle cell anaemia

What is a DNA probe

A short single stranded DNA strand that is complementary to DNA in DNA sample

They can be labelled with flourescent dye to be seen by UV or radioactive markers in photographic film

They locate certain genomes, identify same genes for a genome study and can identify certain alleles

What is genetic engineering ?

Manipulation of DNA sequences and genetic material in an organism to modify their characteristics often involving gene transfer

Overview : obtain required gene

Insert copy of gene into vector

Vector used to insert gene into a host cell

Recipient expresses novel gene

Describe the first stage of genetic engineering

Method A : obtain MRNA via using reverse transcriptase to convert it to cDNA. Add primer and DNA polymerase to make it double stranded

Method B: DNA probe locates gene and cuts out using restriction enzymes

Method C : use restriction endonucleases like ecoR1 to recognise the specific restriction site on the genome (palindromic) making cuts at these points to seperate that fragment from the rest of the DNA and is staggered cut and leaves sticky ends with exposed bases

Desc 2 state of GE

1. Extract plasmid from bacteria or virus

2. Cut plasmid with same restriction endonucleases so they have complementary stick ends

3. DNA from plasmid and dna obtained from gene anneal using polymerase and ligase

Dec stage 3 GE

Vector carrying the gene into the recipient cell

Method A : heat shock so alternate periods of cold 0C and hot 42C in presence of calcium ions.

Ca2+ surround negative charged phospholipids and parts of DNA so make bacterial cell membrane and forging DNA less repulsion

Method B : electroporation so high voltage applied to cell to disrupt membrane and allow transfection by plasmid

Use of agrobacterium tumefaciens

Recombinant plasmid inserted into bacteria ^ this usually infects plant cells so naturally inserts its genome into the host

Some bacteria don’t take it up but those that do are transformed bacteria

What is reproductive cloning

It needs totipotent cells, it is important for genetically identical animals to be made and can be important for producing large numbers of genetically modified animals like goats with spider silk in milk

Desc embryo splitting

1. Collect embryo and sperm from desirable parents and fuse in vitro

2. That zygote divided by mitosis to form small ball of cells

3. Divide embryo into fragments

4. Each small mass of cells placed into uterus of surrogate mother

Can’t see the phenotype before it’s born

Desc SCNT

1. collect an udder cell and egg cell from donor

2. Remove nucleus from both cells

3. Use an electric chock to fuse the empty egg cell and skins nucleus

4. That shock also triggers nucleus to divide by mitosis as if it has just been fertilised causing mitosis

5. The embryo gets placed into a surrogate mothers uterus

The only way to clone an adult which is good as can see phenotype

Not 100% clone as there is some mitochondrial DNA from egg donor left

What is reproductive cloning and examples

Producing clones for some other reason than reproductive purposes.

Aka therapeutic cloning

Stem cells can replace whole organs and tissues eg skin grown in vitro to be a burn area graft

It’s like cloning your own cells so they are identical and don’t cause rejection and is often used for spinal injury

Advs and dis of cloning

Used in scientific research to test effects of medicinal drugs on - avoids humans

Whole herds can have desirable characteristics

Clone endangered animals

A lack of genetic variation

Cloned animals live shorter less healthy lives

Lack of animal welfare : chicken too much meat

How do plants naturally clone themselves ?

They have cells which can differentiate and do this anyway but do it via vegetative propagation:

Leaves: kalanchoe: plantlets on margin, drop off and form new plant

Runner and rhizome:strawberry : horizontal stems that form new roots at a certain point

Sucker: new stem that grows from plant roots and they replace old branch that may die

Bulbs: contain several apical buds to form new plant

Corm: same

Disadvs and advs of natural cloning

Good conditions for parent=good for child

Genetically identical

Fast so can exploit favourable conditions

Only need one parent

No genetic variation

What if environment changes ? - all suffer

So fast that plants may become crowded

What is tissue culture/micropropagation

Makes many clones from just one cutting it takes a small piece of plant the ex plant and uses growth substances to encourage growth and development into new plant

Stages of micropropagation

1. Use sterile forceps to remove growth tip, meristem is used as is viral infection free and sterilise plant with beach or alcohol as bacteria and fungi

2. Place explains into agar containing nutrients like glucose,AA and growth substances like Austin

3. Cells of explant divide by mitosis to form a callus a mass of undifferentiated totipotent cells

4. Callus divides to make more small clumps of undifferentiated cells

5. They differentiate into different types of tissue by moved to different growth media’s

6. Tiny platelets that grow get moved to greenhouse and repotted

Advs and disadvs of artificial cloning of plants

Very fast

Genetically identical

Using meristem so no viral infection

Can grow plants that can’t sexually reproduce

Takes skilled and is labour intensive

Expensive

No genetic variation

Issues with GM with animals + advs

We can breed mice for research

Gene knock out in rats to see if their genes function similar to ours

Animal welfare

Pharming ?

Using GM transgenic animals to produce pharmaceutical products/proteins/products like antithrombin in goat milk

Adv and disadv of pharming

Can produce proteins we need

Some proteins that we can make can’t be done in bacteria as are too large

Animal welfare issue

Food production adv and disadv

Eg salmon we can grow food more rapidly

Outcompete wild populations

Superweed formation that are herbicide resistant weeds

Soya

Resistant to herbicides so only weeds die

Superweed formation

Beta carotene rice + patent

Gene inserted into rice from daffodil that gets a precursor to vitamin A

But if parent put, poor farmers cannot afford and patent means they can’t procure for a period of time

describe the insertion of a DNA fragment into a vector

1. cut vector at a specific site using same restriction enzyme to create complementary sticky ends

2. DNA ligase forms phosphodiester bonds between sugar and phosphate groups on the two strands of DNA joining the sticky ends together

3. Forms recombinant FNA