6.1.3 manipulating genomes

What is a restriction enzyme

Enzyme that cuts DNA at specific recognition sites which produces DNA fragments

Where are Restriction enzymes normally found

Bacteria

what is a recognition site ?

Specific base sequence

Why are recognition sites palindromic ?

Read the same backwards and forwards

What are the types of cuts made by restriction enzymes

Sticky ends

Blunt ends

what is the difference between sticky and blunt ends

Sticky ends → there’s a overhang of DNA bases

Why are restriction enzymes used on Non - coding genes?

There are no mutations in Non- coding genes base sequence → easier to predict results

Function of non- coding genes

Control transcription by switching genes on and off

what is the function of Gel electrophoresis

Separate the DNA fragments as DNA is invisible

Why does DNA move through the gel when an electric current is applied?

DNA contains PO₄³⁻ anion on nucleotide this is attracted to the Positive electrode ( Anode)

what is the colour of DNA

Colourless

Control variables when using enzyme

Use the same restriction enzyme → creates same complementary sticky ends

Function of the gel in gel electrophoresis

Creates a matrix mesh that keeps large DNA fragments at top ( cant travel through mesh) and small at the bottom

which DNA fragments move faster and further in Gel electrophoresis

Small fragments

Function of Buffer ( Salt) solution in gel electrophoresis

maintains charge helps the current move

why are DNA fragments / banding pattern different?

Different alleles → different specific recognition sites

Different number and sizes of DNA fragment are produced

DNA fingerprinting ( Image)

Limitations with gel electrophoresis

DNA fragments can get stuck in the gel

its not 100% could be related ( Identical twins)

Function of PCR in DNA fingerprinting

Used to amplify the DNA so there is enough to be analysed

DNA profiling method

Extract DNA

DNA fragments are placed into agarose gel

apply electric current

DNA moves to positive anode

smaller fragments move further and faster

compare banding pattern with DNA sample to see if there similar.

Function of PCR

Amplify DNA fragment for further study or processing

What does PCR stand for

polymerase chain reaction

Uses of DNA profiling

Forensics

diagnosis of genetic disorders

What are the steps of PCR

Denaturation → separate double helix

Annealing → Primers bind to the start of the target DNA

Synthesis → Building the new strand

PCR what are primers

short pieces of DNA that are complimentary to the bases of the fragment

PCR what happens during denaturation?

DNA mixture heated to 95°C → breaks Hydrogen bonds

separates DNA strands

Why can many cycles of PCR happen without needing new enzymes

DNA polymerase doesn’t denature at high temp

PCR what happens at annealing stage

55°C temp of reaction mixture

allows primers to bind to start of strands

PCR what happens at the synthesis stage

72°C temp allows DNA polymerase to work

DNA polymerase joins DNA nucleotides→ forms template strand using complimentary base pairings → forms 2 new complimentary strands

what type of DNA polymerase is used in PCR

Taq DNA polymerase → doesn’t denature at high temperatures

human does denature at 72°C

what molecules are required for PCR

 DNA template

primers

nucleotides

DNA polymerase

Limitations of PCR

Expensive

Takes a long time

what molecules are required for chain termination method

DNA polymerase

Primer

DNA sample

nucleotides

Fluorescently-labelled modified nucleotide / terminator base

Chain termination method first step

Mixture of required molecules is added to separate tubes

chain termination method second step

Tubes undergo PCR

strands produced are different lengths → terminator bases terminates the strand ( cant extend strand)

terminator nucleotide is added on different points

why do fluorescently-labelled modified nucleotides terminate the strand

No bases can be added after

Hydrogen instead of hydroxyl group on carbon 3 so cant form phosphodiester bond with next nucleotide

methods to read data in DNA sequencing

Gel electrophoresis

Capillary sequencing

electrophoresis method in DNA sequencing

electrophoresis separate DNA fragments

shine under UV light / Fluorescent tags

Read from top to bottom ( smaller fragments are near top / move furtherest)

How can DNA be sequenced ?

chain termination method

Advantages of using chain termination method for DNA sequencing ?

allows for massive parallel sequencing

sequence many DNA at same time

what are the solutions that are used in electrophoresis

Buffer solution → helps the current move down due to ions

Loading dye → increases contrast ( Visualise Patterns / see fragments)

why is temp 72°C - 75°C used for synthesis stage

Taq polymerase optimum temp is ( named temp)

Taq polymerase can withstand high temp

Fastest rate of reaction

In electrophoresis what does the sample have to be

Has to be negatively charged

( protein denature)

What is synthetic Biology ?

The design and construction of new biological systems or redesign

Explain how gene sequencing can be used to compare genomes between different species

The two organism are likely to be closely related as they have a similar base sequence

what is the difference between Bioinformatics and Computational biology?

Bioinformatics → Storing data sets, making data base

Computational → analysis of large data sets

How is Gene sequencing used in production of protein

Determine order of Bases

Determine order of amino acids

Primary structure of protein

How is Bioinformatics used in production of protein

Stores large amounts of data

Produces database of gene sequencing and protein structure

used to share information → Format is universal

How is Computational Biology used in production of protein

Analysis of large data sets

predict amino acid sequence

modelling protein structure + function

Function of primers in PCR

bind to template strand → allows DNA polymerase to work

Uses of GE

GE to increase crop yield → disease, herbicides and pesticides resistance

GE of livestock → pest and disease resistance

GE of bacteria → produce insulin

GE process ?

Use a restriction enzyme to cut desired gene out of cell → produces sticky ends

Cut plasmid with the same Restriction enzyme at Marker gene → Gene inserted into a plasmid of bacteria using DNA Ligase

Insert recombinant gene into Host cell

increase rate with electroporation.

Function of DNA ligase

Forms phosphodiester → sticks desired gene and plasmid

differences and Similarities between DNA polymerase and ligase

similarities → Both form Phosphodiester bonds, both catalyse condensation reactions, both made from DNA

differences → DNA polymerase joins individual nucleotides in DNA replication

Function of electroporation / Heat shock in GE

Make the bacterial membrane more permeable → increases rate of plasmid up take from bacteria

Advantages of using bacteria used in GE?

Bacteria replicate rapidly

easy to maintain & produce in a Lab

(GE) How to increase efficiency of DNA uptake

place bacteria in a solution of Ca²⁺ → neutralise the negative charge on cells outer membrane so DNA can enter cell

Electroporation

in GE why are bacteria kept cold

stabilise the cell membrane → closes phospholipid bilayer pores

What is meant by DNA sequencing ?

Determining the sequence / Order of nucleotides in DNA

How are virus tracked over time for mutations ?

Bioinformatics

Why would DNA profiling be used in Agriculture

Prevent inbreeding in selective breeding → reduces gene pool

DNA profiling what does 2 identical bands mean for 1 individual

They are homozygous for the trait

In GE what is a marker and give example?

marker → allows the scientists to test which bacteria have taken up the recombinant plasmid ( millions of bacteria)

Antibiotic resistance gene → Grow on agar containing antibiotics → only bacteria who have taken up plasmid successfully will survive

Describe a PCR graph

Exponential Growth

free nucleotides are limiting factor → graph plateaus

How to work out number of Nucleotides / length of a gene

number of amino acids x 3 ( there are 3 nucleotides in codon)

How to read sequence of bases in DNA (Roche pyrosequencing) ( Image)

State two development that has lead to a increase in the rate of DNA sequencing ?

WGS ( Whole genome sequencing)

Massive parallel sequencing

How can DNA sequencing allow the sequence of amino acids in polypeptide to be predicted ?

Determine the Order of bases that code for the amino acid sequence

Codons code for each amino acid

Explain how How occurrence of disease is example of Artificial selection

Shows inbreeding → small gene pool

What section of DNA is used when profiling humans

Non- coding DNA

Why are only selected sections of non-coding DNA used when profiling humans ?

Humans have similar genomes

coding sequence doesn`t produce unique profile

sections of non coding DNA contain variable numbers of repeating sequences

How to work out number of fragments in PCR

Log (x) → if they want in Log value

What are the advantages of GM organisms ( plants)?

Reduces concentration of chemical pesticides used → harm environment

designed to be more nutritious

Negative ethical issues about using GM organisms ( Plants)

Encourages monoculture → reduces biodiversity

GM plant could interbreed with wild plants → superweeds ( resistant to herbicides)

What is meant by pharming ?

using GM organisms to make drugs

Pharming ( Image)

Advantages and advantages of using Pharming

A: Large quantities of Drug → more available to people

D: Potential harmful side effects

Disadvantages of using GM pathogens

could potentially be used in Biological warfare

Lead to a outbreak of disease

Advantages of using GM pathogens

previously Untreatable diseases can be treated

What is meant by Technology Transfer?

Scientists sharing knowledge, tech and skills of GM products

What is a patent?

Companies obtaining legal protection and ownership over their product

Advantages of patents?

Owners will get more money from selling product → encourages competition for new beneficial GE ideas

Disadvantage of Patents?

Farmers from poorer countries cant afford patented GM seeds → legally cant grow crop / seeds

How is gene therapy used on Homozygous recessive conditions ?

Add a working dominant gene

How is gene therapy used Dominant conditions ?

silence the Dominant allele → insert DNA using vector ( altered virus)

What are the types of Gene therapies?

Somatic therapy

Germ line therapy

What is somatic therapy?

Altering alleles in body cells

Disadvantage of Somatic therapy

Doesn`t alter Sex cells → Offspring could still inherit disease

Effects may be short- lived

What is Germ line therapy?

Altering alleles in Sex cells → offspring will not inherit disease

Positive ethical issues of Gene therapy

Prolong the lives of people with terminal genetic disorders

Decrease number of people that suffer from genetic disorder → economical fewer people require treatment

Allow carrier of Genetic disorders to conceive baby without disorder

what are the Negative ethical issues of gene therapy?

Gene therapy is expensive → health service could be spent on better treatments

Could be used in ways other then medical treatment → cosmetics

risk of overexpression of genes

what are the Disadvantages of gene therapy

inserted allele could get overexpressed

Body could identify vectors as foreign and immune response against them

Difficult to get allele into specific body cells ₂

What is meant by Gene therapy

Altering the alleles inside cells to cure genetic disorders

Disadvantages of Chain-termination method ?

Cant sequence entire genomes only DNA fragments up to 750 bp

Differences between Synthetic biology and GE

GE involves direct transfer of DNA between organisms

Synthetic biology → creates DNA

Process of creating GM plant ( resistance)

Use restriction enzyme to cut out desired gene from bacteria

use the same RE to cut out plasmid from vector → creates complementary sticky ends

Insert gene into plasmid, use DNA ligase → forms phosphodiester bonds → creates recombinant plasmid

insert recombinant plasmid into bacteria → transfer recombinant plasmid DNA into plant cells

Use marker genes ( antibiotic / fluorescent gene) to show which cells have successfully taken up gene

electroporation → get plasmid inside cell

How can mRNA be altered so it can be used in GM in bacteria ?

Use reverse transcriptase to convert mRNA → cDNA

Use DNA polymerase to make double stranded DNA

advantages of using mRNA instead of DNA during GM?

mRNA has been spliced → contains Exons only

DNA contains introns ( non- codin

How can Scientists use PCR to compare growth of bacteria on tissues

Extract DNA from both sources ( named)

compare rate of amplification after 30 cycles of PCR