Biology- manipulating genomes

How is DNA replicated on a large scale

PCR

What does PCR stand for

Polymerisation chain reaction

In PCR what’s the first step and why

DNA is heated to 96 degrees to denature the strands

What happens in the second stage of PCR and why

The reaction is cooled to 60 degrees allowing primers to anneal to the DNA strands

What happens in the final stage of PCR and why

The reaction is heated to 72 degrees allowing the nucleotide chain to be extended using DNA polymerase

How many times is PCR usually repeated

25-35 times

What’s gel electrophoresis

A technique used to separate DNA fragments by size

What are introns

Large non-coding regions of DNA

What are exons

The DNA that codes for proteins

What are VNTR’s

Sequences of DNA 20-50 base pairs long that are repeated 50-100 times

What are VNTR’s also known as

Minisatelites

What are STR’s

Sections of DNA 2-4 base pairs long repeated 5-15 times

What are STR’s also known as

Microsatelites

Why are exons not often used in genetic profiling

Because the nucleotide differences are too small

What are the 5 key components in PCR

DNA polymerase, primers, free nucleotides and a DNA sample

What’s different about taq DNA Polymerase

It can withstand the temperatures used in PCR due to its thermal stability

How does gel electrophoresis work

DNA is negatively charged due to its phosphate group and so moves along the agarose gel towards the positively charged anode, different DNA fragments have a different number of repeats and so travel different distances along the gel

What are 3 key differences between gel electrophoresis and TLC

1. TLC separates by solubility rather than size

2. TLC separates non-charged particles

3. Electrophoresis requires an electric current TLC doesn’t

Why do smaller fragments travel further through the agarose gel

They can fit through the pores in the agarose gel more easily than larger fragments

Where is the CFTR protein located

The integral membrane protein

What’s the difference between a normal CFTR and mutant CFTR channel

In a mutant CFTR channel chloride ions aren’t transported outside the cell, causing the mucus to become thicker and stickier due to a lower water content

What are some uses of DNA profiling

In forensic science, paternity tests and species identification

Who was Frederick Sanger

The first scientists sequence nucleic acids from viruses and bacteria

What did Sangers discovery lead to

Capillary tube electrophoresis

What allowed the human genome project to be completed ahead of the initial timeframe

The introduction of new faster and more powerful computers

What’s DNA sequencing

The technique of determining the precise corded of nucleotides within a DNA molecule

What are terminator bases

Nucleotides tagged with a fluorescent marker that stop DNA polymerase working

What are the 5 steps involved in genome sequencing

1. The DNA is mixed with a primer, DNA polymerase, an excess of free nucleotides and terminator bases

2. The mixtures is put into a thermal cycler

3. Free nucleotides are added to the DNA strands but when a nucleotide with a terminator base is added the DNA can’t be extended any further

4. Many copies of different lengths of DNA are produced with different coloured tags at the end

5. The DNA fragments are separated by gel electrophoresis and a laser is used to record the tag colour

How does Next-generation sequencing work

Sequencing occurs on a flow cell instead of in a capillary tube allowing millions of DNA fragments to be replicated in situ using PCR forming clusters of identical DNA fragments

What’s bioinformatics

the development of the software and computing tools needed to organise and analyse raw biological data

How can bioinformatics be used in relation to an organisms genome

Using a high-powered computer a database can be formed sequencing an organisms genome allowing comparisons between organisms

What’s computational biology

Using the data produced by bioinformatics to build theoretical models of biological systems

How is computational biology used in relation to an organisms genome

It can be used in the analysis of the data from sequencing the billions of base pairs in DNA for working out the 3D structure of proteins

What’s genomics

The field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes

What are some applications of analysing the genomes of pathogens

Allowing doctors to identify the source of an infection or identify antibiotic-resistant bacteria and allowing scientists to identify regions of the genome that may be useful in the development of new drugs

What’s DNA barcoding

Identifying particular sections of the genome that are common to all species but vary between them

What region of genome is used for DNA barcoding in animals

A section of the mitochondrial DNA in the gene cytochrome c oxidase

What organisms is DNA barcoding not suitable for and why

Fungi and bacteria as there have not been suitable regions identified

how can scientists calculate how long ago two species diverged from a common ancestor

By calculating the basic mutation rate of DNA from two different organisms

What’s proteomics

The study and amino acid sequencing of an organisms entire protein complement

How do scientists know that genes can code for multiple different proteins

Because there are a smaller number of coding genes in the human genome than proteins

What are spliceosomes and what do they do

They’re enzyme complexes that join together exons to form mature functional mRNA

How do spliceosomes contribute to different phenotypes

They can join the same exons in a variety of different ways producing different versions of functional mRNA

How can protein modification help explain the relationship between genotype and phenotype

Proteins can be modified by other proteins after being synthesised allowing them to be lengthened or shortened and even form different proteins

what’s synthetic biology

the design and construction of novel artificial biological pathways, organisms or devices or the redesign of existing natural biological systems

What different techniques does synthetic biology include

Genetic engineering, using biological systems in industrial contexts, synthesising new genes to replace faulty ones and synthesising new organisms

What’s genetic engineering

when the genes from one organisms are transferred into another organisms using a vector in such a way that the genes are expressed

What are the key steps in the genetic engineering process

Identifying the desired gene, isolating the plasmid, isolating the gene of interest (cutting this and the plasmid using the same restriction endonuclease), inserting the gene into the plasmid using DNA ligand, insert the plasmid into bacterium and reproducing the GMO’s

What’s a transgenic organism

An organism with nucleotide sequences from a different species

What are restriction endonucleases used for

To cut genes at specific base sequences

What’s the function of DNA ligase

To join together the cut ends of DNA by forming phosphodiester bonds

What’s the function of reverse transcriptase

To build double stranded DNA from single stranded RNA

What’s a vector

Something used to deliver DNA fragments into a cell

What are 3 common vectors

Plasmids, viruses and liposomes

How is vector success monitored

Using marker genes

What are 2 common marker genes

Antibiotic resistance or fluorescence

What’s electroporation

Using small electric currents to make bacterial membranes permeable allowing plasmids to enter the cell

Why must the electric current be controlled during electroporation

To avoid permanent damage to the cell membrane

What are the 3 main methods of producing DNA fragments

1. Direct synthesis of DNA from a proteins primary structure

2. Converting mRNA to cDNA

3. Isolating the gene from its DNA sequence

What enzyme is used to form cDNA

Reverse transcriptase

What type of ends form when restriction endonucleases cut at the same point on both sugar-phosphate backbones

Blunt ends

What type of ends form when restriction endonucleases cut one sugar-phosphate backbone longer than the other

Sticky ends

Why do sticky ends bond together more easily than blunt ends

Because hydrogen bonds can form between the exposed bases on the identical sticky ends

What’s ligation

Sticking 2 strands of DNA together

What method is used to identify whether bacterial plasmids contain the desired gene

Replica plating

how are marker genes used to indicate whether a DNA fragment has been successfully inserted

The DNA fragment is inserted into a marker gene and if this is done successfully the marker gene won’t function

What’s electrofusion

Tiny electric current are applied to the membranes of two different cells to fuse the cell and nuclear membranes of the two cells

What must be inserted along with a marker gene

Promoters to ensure that the gene is transcribed

Why is it easiest to access bacterial DNA

There are no histones so the DNA is easily accessible

What are some limitations of using bacterial DNA in genetic engineering

There are no introns and therefore no mRNA splicing

What’s an example of genetic modification in plants

Golden rice wheee beta carotene was inserted into rice

What are the ethical concerns associated with genetic modification of microorganisms

GM microorganisms could be used in biological warfare

What are the benefits of GM microorganisms

The production of medicines, antibiotics and increasing the food supply for the growing population

Explain how soya beans have been genetically modified

A gene has been inserted into soya beans that produces the Bt protein which is toxic to many pests that attack the plant increasing crop yield

How has patenting affected the usefulness of GM crops

Many crops including GM soya beans have been patented making them more expensive and less accessible to people in less economically developed countries the seeds must also be re bought every year and cannot be replanted

What are micro injections and how are they used in GM

DNA covered in gold can be injected into organisms introducing new genes to animal DNA

What are examples of GM animals

Swine fever-resistant pigs and faster-growing salmon

What’s pharming

the production of human medicines in animals

What are the 2 main ways pharming is used

1. Creating animal models

2. Creating human proteins

What are ethical issues associated with pharming

Is it right to put human genes into animals? Is welfare compromised during the production of genetically modified animals?

What’s somatic gene therapy

Replacing a mutant allele with a healthy allele in the affected somatic cells

What’s a limitation of somatic cell gene therapy

It’s only a temporary treatment because somatic cells have a limited life and are replaced from stem cells with the faulty allele

What’s germ line cell therapy

treating a disease by inserting a healthy allele into the germ cell

Why is germ line cell therapy not practised

Due to the ethical and medical concerns around intervening with germ cells

What’s gene therapy

Fixing cellular problems by replacing faulty genes in somatic or germ line cells by inserting a normal one

What’s a germ cell

A pre-gamete cell involved in reproduction