Unit 5- Genes and DNA (Ch14-16)

What is the genetic code?

Base triplets in mRNA which code for specific amino acids

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Give 3 features of the genetic code.

1) Degenerate - most amino acids have more than one codon. 2) Non-overlapping- each base in sequence is only read once. 3) Universal- the same codon codes for the same amino acid in all organisms (with a few minor exceptions)

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What is a codon?

A sequence of 3 nucleotide bases on mRNA

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Some codons code for no amino acids. What are these called, how many are there, and what do they do?

Stop codons, 3, mark the end of a polypeptide chain

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What is RNA?

A polymer made up of repeating mononucleotide sub-units.

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Each nucleotide is made up of:

A pentose sugar (ribose), one of the organic bases (AUCG), a phosphate group

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What are the two types of RNA important in protein synthesis and what do they do?

messenger RNA (mRNA)- acts as a template upon which proteins are built. Transfer RNA (tRNA)- lines up amino acids on the mRNA template during protein synthesis

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What happens once mRNA is formed?

it leaves the nucleus via nuclear pores and enters cytoplasm where it associates with ribosomes

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What is mRNA's structure, and how is it suited to its function?

A long strand arranged in a single helix- it possesses the correct sequences of many triplets of organic bases that code for specific polypeptides, itts easily broken down and hence only exists while it is needed to manufacture a given protein

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What is tRNA's structure, and how is it suited to its function?

A single-stranded chain folded into a clover-leaf shape, with one end of the chain extending beyond the other. At extended end, amino acids can easily attach, at opposite end is an anticodon which pairs with complementary codon on mRNA

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Arrange DNA, mRNA and tRNA in order of a) size (from smallest to largest), b) stability (from least to most)

a) tRNA-->mRNA-->DNA b) mRNA-->tRNA-->DNA

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What happens during transcription?

an mRNA copy of a gene is made in the nucleus

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Describe the process of transcription (Step 1)

1) DNA helicase acts on specific region of DNA molecule to break H bonds, unwinding the double helix and exposing the bases in that region.

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Describe Step 2 and 3

2) RNA polymerase moves along the template strand, causing nucleotides on this strand to join with complementary nucleotides from pool within nucleus. 3) When polymerase reaches a stop codon, it detaches and the production of pre-mRNA is complete.

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Why will only 12 bases on DNA be exposed at any one time?

As RNA polymerase adds nucleotides one at a time to build a strand of pre-mRNA, the DNA strands rejoin behind it

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What is splicing?

When the pre-mRNA is edited by removal of non-functional introns (sections of DNA that don't code for amino acids)

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What cells does splicing occur in?

Eukaryotic cells

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Once the introns have been removed, what happens to the remaining exon sections?

They can be recombined in a variety of combinations- a single gene can code for up to a dozen different proteins, depending on the order in which the exons are recombined

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Where does translation occur?

Ribosomes in the cytoplasm

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What happens during translation?

amino acids are joined together to make a polypeptide chain, following the sequence of codons carried by the mRNA

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Describe the process of translation. (step 1 + 2)

1) Ribosome attaches to the starting codon at one end of mRNA molecule. 2) tRNA molecule (which is carrying an amino acid), with a complementary anticodon sequence moves to the ribosome and pairs with the sequence on the mRNA

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Describe step 3 and 4

3) a tRNA molecule with a complementary anticodon pairs with the next codon on the mRNA, carrying another amino acid. 4) the ribosome moves along the mRNA bringing together two tRNA molecules at any one time

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Describe step 5, 6 and 7

5) The 2 amino acids on the tRNA are joined by a peptide bond- involving an enzyme and ATP 6) the ribosome moves onto 3rd codon in mRNA sequence 7) 1st tRNA is released from its from its amino acid and is free to collect another from pool within cell

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When will the ribosome, mRNa and last tRNA separate?

When the ribosome reaches a stop codon, and polypeptide chain is complete

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How many amino acids can be linked per second?

15

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What happens to the polypeptide chain? (3 things)

1) coiled/folded to produce secondary structure. 2) secondary structure is folded further to produce tertiary structure 3) different polypeptide chains, along with any non-protein groups are linked to form quaternary structure

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What is a mutation?

Any change to the quantity or structure of the DNA of an organism

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What is a gene mutation?

Any change to one or more nucleotide bases in DNA

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GIve two different types of mutation.

Substitution of bases or deletion of bases

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What is the substitution of a base?

When a nucleotide in a DNA molecule is replaced by another nucleotide that has a different base

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What are the 3 possible consequences of a base substitution?

1)a nonsense mutation- base change results in formation of a stop codon (production stopped prematurely) 2)a mis-sense mutation- base change results in a different amino acid being coded for 3)a silent mutation- same amino acid still coded for

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What is the deletion of a base?

When a nucleotide is lost from the normal DNA sequence

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What is the result of a deletion?

A 'frame-shift' because the reading frame that contains each 3 letters of code is shifted to the left by 1 letter

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What are the causes of mutations? (2)

can occur spontaneously during replication OR be the result of mutagenic agents e.g. high-energy radiation or chemicals that interfere with transcription

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A mutation is likely to matter when...

its an addition/deletion, its near the start of a gene, the position/structure of mutated amino acid is important (e.g. if its on an active site), if the protein containing mutation is important, if its in germ cells (gametes)

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A mutation is less likely to matter if...

its a substitution, its on the 3rd base of a codon, its in a gene that isn't expressed in the cell, its in non-coding DNA, when its in somatic cells (body cells)

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Why do most cells divide constantly?

To ensure that dead cells are replaced.

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In normal cells, the rate of division is tightly controlled by what two things?

1) proto-oncogenes- stimulate cell division. 2) tumour suppressor genes - slow cell division

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Desrcibe how proto-oncogenes work

Growth factors attach to a receptor protein on cell-surface membrane. Via relay proteins in cytoplasm, they 'switch on' genes necessary for DNA replication

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What can a gene mutation in a proto-oncogene do?

Cause them to mutate into oncogenes.This can permanently activate receptor so that cell division is switched on 24/7 (even in absence of growth factors), or may code for a growth factor that is produced in excess

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Describe how tumour suppressor genes work

by maintaining normal rates of cell-division and preventing tumours from forming

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If mutated...

...they are inactivated, and therefore cell division increases.

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What happens to the mutated tumour suppressor genes?

Most die, but if they survive they can form tumours by cloning themselves

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What is a totipotent cell?

A cell which can mature into any body cell

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During the process of cell specialisation, only some of the genes are expressed- hence only part of the DNA of a cell is transcribed and translated into proteins. How are genes prevented from expressing themselves? (2 ways)

1) preventing transcription and hence preventing the production of mRNA. 2) Breaking down mRNa before its genetic code can be translated

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What is a multipotent cell? (give example)

cells that can become a wide range of cells, but not any cell e.g. adult stem cells

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Where are adult stem cells found?

In the inner lining of the small intestine, in the skin, in our bone marrow

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Under certain conditions, adult stem cells can develop into other types of cell. Give an example of this.

E.g. red blood cells can be produced from a type of stem cell in the bone marrow. Red blood cells contain lots of haemoglobin and no nucleus - stem cell develops into a cell in which genes for haemoglobin production + removal of nucleus are expressed

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What are embryonic stem cells a type of?

Totipotent cells

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Describe the process of using embryonic stem cells in medicine (step 1-4)

1) early embryo is cultured in a nutrient medium 2) outer layer collapses and inner cell mass is freed from embryo 3) chemicals are added to break up the cell mass into smaller groups 4) each group grows a colony

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Describe step 5+6

5) Special differentiation factors are added to colonies in separate containers 6) the differentiated cells are transferred to damaged tissues

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What is the stem cell situation in plants?

Mature plants have many totipotent stem cells found in areas where the plant is growing e.g. roots and shoots

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What are these plant stem cells used for?

To grow plant organs (e.g. roots) or whole new plants in vitro

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What is growing plant tissue artificially known as?

A tissue culture

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Describe the process of growing a tissue culture (steps 1-3)

1) a single totipotent stem cell is taken from a growing area on a plant 2) the cell is placed in some sterile growth medium containing nutrients and growth factors 3) the plant stem cell will grow and divide into a mass of unspecialised cells-

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Describe step 4

4)If conditions are suitable, the cells will mature into specialised cells, either to form a plant oran or an entire plant depending on what growth factors are used

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Outline 2 ethical issues surrounding stem cell use

1) obtaining stem cells from IVF involves the destruction of an embryo that could become a fetus if placed in a womb 2) some people believe that from the moment of fertilisation the individual formed has the right to life and shouldn't be destroyed

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So what alternatives can we use to avoid these objections?

1) use adult stem cells (BUT these are only multipotent), 2) obtain stem cells from unfertilised embryos (made from egg cells that haven't been fertilised by sperm), because they won't produce a fetus if placed inside the womb

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What are transcriptional factors?

Specific molecules that move from the cytoplasm into the nucleus and stimulate the gene for transcription to begin

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Each transcriptional factor has...

...a site that binds to a specific region of the DNA in the nucleus.

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When a gene is not being expressed, it means...

...the site on the transcriptional factor that binds to the DNA is blocked by an inhibitor molecule, which prevents the transcription factor from binding to DNA and hence prevents transcription and polypeptide synthesis

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What is SiRNA?

Small interfering RNA

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What does breaking down mRNA do?

prevents gene expression before its genetic code can be translated into a polypeptide

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Describe how it works (step 1 +2)

1) an enzyme cuts the large double-stranded molecules of RNA into smaller sections called siRNA 2) one of the 2 siRNA strands combines with an enzyme

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Step 3, 4 and 5

3) the siRNA molecule guides the enzyme to mRNA molecule by pairing up its bases with complementary ones on a section of mRNA molecule 4) once in position, the enzyme cuts the mRNA into smaller sections 5) mRNa no longer capable of being translated

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Describe 2 possible scientific/medical uses of siRNA

1) May be possible to block genes that cause disease 2) could be used to identify the role of genes in biological pathway (e.g. siRNA that blocks a particular gene could be added to cells, and by observing effects we could determine what the role is)

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How do hormones such as oestrogen switch on a gene and hence start transcription?

By binding with a receptor on the transcriptional factor, which releases the inhibitor molecule

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Describe the 5 step process (steps 1 + 2)

1)Oestrogen is a lipid-soluble molecule and therefore diffuses easily through the phospholipid portion of cell-surface membrane 2) Once inside the cytoplasm, oestrogen combines with a site on a receptor molecule of the transcriptional factor

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Describe step 3

3) By combining with the site, the oestrogen changes the shape of the receptor molecule. This releases the inhibitor from the DNA binding site on the transcriptional factor

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Describe step 4 and 5

4) The transcriptional factor can now enter the nucleus through a nuclear pore and combine with DNA 5) This simulates the transcription of the gene that makes up the portion of DNA

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Explain how knowing that cancer can be caused by acquired mutations affects its prevention.

1) protective clothing for people who work with mutagenic agents 2) sunscreen when skin is exposed to UV radiation 3) vaccination- some acquired cancers are caused by viruses e.g. HPV has been linked to cervical cancer

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Explain how knowing that cancer can be caused by acquired mutations affects its diagnosis

High-risk individuals can be screened for cancers that the general population aren't normally screen for- or they can be screened earlier + more frequently. This would lead to earlier diagnosis (b4 symptoms appear), which increases chance of recovery

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Explain how knowing which specific mutation a type of cancer is usually caused by affects its diagnosis. Give example.

More sensitive tests can be developed, which can lead to earlier and more accurate diagnosis, improving the chances of recovery. e.g. there's a mutation in the RAS proto-oncogene in around half of all bowel cancers

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Explain how knowing which specific mutation a type of cancer is usually caused by affects its treatment (Give 1 of 3 ways)

1) treatment can be different for different mutations e.g. breast cancer caused by mutation of the HER2 proto-oncogene can be treated with Herceptin

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What does Herceptin do?

Binds specifically to the altered HER2 protein receptor and suppresses cell division and tumour growth - NOTE - this only works with the HER2 mutation

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Give the other 2 ways

2)the agressiveness of the treatment can differ e.g. if mutation known to cause a fast-growing cancer, can remove larger areas of tumour and surrounding tissue/use higher doses of radiotherapy 3) gene therapy may be able to treat it

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Give example of how gene therapy may be able to treat cancer caused by a known mutation

e.g. if we know its caused by inactivated tumour suppressor genes, it could be used to provide working versions of these genes

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Explain how knowing cancer can be caused by hereditary mutations can affect the prevention of cancer (2 ways)

1) Those with a family history of cancer could avoid gaining extra acquired mutations by avoiding mutagenic agents 2) DNA could be analysed to see if they carry specific mutation, and preventative surgery can be done

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Explain how knowing cancer can be caused by hereditary mutations can affect the diagnosis of cancer

Screening, or increased/earlier screening can lead to early detection and increased chances of recovery

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Explain how knowing genetic disorders can be caused by hereditary mutations can affect their diagnosis

Person can have their DNA analysed to see if they have the mutation or if they are a carrier- can also help figure out if any children they have are at risk

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Explain how knowing the specific gene genetic disorders can be caused by can affect their treatment (3 ways)

1) Gene therapy- e.g. treatment of CF 2) treatment can vary for different mutations- e.g. diff. Huntingdon's mutations affect time of onset and hence symptom treatment options 3) Early diagnosis can affect treatment options

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Give an example of the 3rd way

Sickle cell anaemia can be diagnosed at birth, and treatments that relive symptoms and work to avoid complications can be given straight away

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Explain how knowing the specific gene genetic disorders can be caused by can affect their prevention

Carriers or sufferers can undergo preimplantation genetic diagnosis during IVF to prevent any offspring having the disease. Embryos are produced by IVF, screened for mutation and only those lacking the mutation are implanted in the womb

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Outline the 5 stages involved in transferring a gene and cloning

Isolation, insertion, transformation, identification, growth/cloning

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Isolation of a gene uses either:

a) reverse transcriptase b) restriction endonuclease

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Describe the isolation of a gene using reverse transcriptase (1-3 out of the 5 steps)

1) a cell that readily produces a protein is selected 2) these cells have large quantities of relevant mRNA, which is extracted 3) reverse transcriptase used to make DNA from RNA (known as complementary DNA- cDNA)

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Steps 4+5

DNA polymerase is used to build up complementary nucleotides on cDNA template to form other strand of DNA 5) double strand of dNA is formed, containing the required gene

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What does restriction endonuclease do?

Cuts double stranded DNA at a specific sequence of bases

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When do blunt ends occur?

When the cut occurs between 2 opposite base pairs

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When do sticky ends occur?

When DNA is cut in a 'staggered fashion'- leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases. ( a straight cut through opposite base pairs)

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What is a 6 base-pair palindromic base sequence?

The two sequences of unpaired bases that are opposite to one another, which remain after DNA is cut in staggered fashion. (If complement of one sequence read backward, reads the original sequence)

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What is the importance of sticky ends?

We can combine DNA of one organism with DNA of another, provided same restriction endonuclease is used

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Explain this further.

If same endonuclease is used to cutDNA, then all fragments produced will have ends complementary to one another. This means that the single-stranded end of any one of the fragments can be joined to the single-stranded fragment of any other

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What can be used to then permanently join the two DNA strands?

DNA ligase

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Why do different endonucleases cut at different specific recognition sequences?

shape of recognition sequence must be complementary to enzyme's active site

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Once DNA has been isolated, the next stage is to insert it into a vector. Give two types of vectors that could be used. (note the following flashcards are on in vitro NOT in vivo)

Plasmids or bacteriophages

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Describe the 3 step process

1) DNA fragment inserted into vector DNA 2) vector DNA cut using same restriction endonuclease as target gene, so sticky ends are complementary and they join up. 3) DNA ligase used to join sugar-phosphate backbone of two sections of DNA

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The next step is for the vector to transfer the gene into the host cells. Describe how this can be done if a) a plasmid vector is used and ....(see next card)

a) host cells made permeable by placing in ice-cold CaCl solution b) plasmids added + mixture is heat-shocked (heated for 1-2mins at 42C), encouraging cells to take up plasmids.

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....b) a bacteriophage vector is used

b) bacteriophage infects host bacterium by injecting DNA into it. The phage DNA then integrates into bacterial DNA

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Identification is the next step. Outline how marker genes are used to identify transformed cells. (3 steps)

1)Marker genes inserted into vectors at the same time as the gene to be cloned. 2)Host cells are grown on afar plates + each cell divides + replicates its DNA 3) transformed cells will produce colonies containing cloned gene + marker gene

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Give 3 things the marker gene could code for, and outline how this would be identifyable

1) Antibiotic resistance- grow on agar plates containing specific antibiotic (only transformed cells survive) b) Fluorescence- agar plate is placed under a UV light (only transformed cells fluoresce) 3) enzyme production

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Explain/give example of how enzyme production can help to identify a transformed gene

e.g. lactase will turn a particular colourless substrate blue. Required gene is transplanted into gene which makes lactase, hence if plasmid contains required gene lactase WON'T be produced. (UNtransformed will turn substrate blue)

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What is the final step of in vivo?

Identified transformed cells are allowed to grow more, producing many copies of the required cloned gene

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What does in vivo DNA cloning involve?

The polymerase chain reaction (PCR)

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What can PCR do?

Make millions of copies of a fragment of DNA in just a few hours.

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Define a) DNA polymerase b) primers

a) enzyme that creates new DNA strands by joining together nucleotides b) short sequences of nucleotides that have a set of bases complementary to those at one end of each of 2 DNA fragments

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What is a thermocycler?

a computer-controlled machine that varies temperature precisely over a period of time

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What 3 main steps does PCR involve?

Separation of the DNA strand, addition (annealing) of the primers, and the synthesis of DNA

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Describe the first stage (separation of the DNA strand- 2 steps)

1) The DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler 2) The temperature is increased to 95C, causing the two strands of the DNA fragments to separate

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Describe the second stage (addition (annealing) of the primers)

The mixture is cooled to 55C, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment

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What do the primers do? (2 things)

provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. They also prevent the two separate strands from simply rejoining.

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Describe the third stage (the synthesis of DNA- 3 steps)

1)The temp is increased to 72C 2)free DNA nucleotides lined up alongside each template strand- new complementary strands are formed. 3)It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.

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How many new copies of the fragment of DNA are formed when one cycle of PCR is complete?

Each PCR doubles amount of DNA

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Give 2 advantages of in vitro (PCR)

1) very fast- valuable when only small amount of DNA available 2) doesn't require living cells- complex culturing techniques aren't needed

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Give 2 disadvantages of in vitro (PCR)

1)It requires a very pure sample as any contaminant DNA would also be multiplied. 2) At any one time, 20% of the DNA cloned is copied inaccurately although modern techniques have now improved.

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Give 2 advantages of in vivo

1)no risk of contamination as the restriction endonuclease can match the ‘sticky ends’ together 2) • It is very accurate as the DNA copied has very few, if any, errors. 3) • Cuts out specific genes- so very precise, doesn’t copy whole DNA sample

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Give 2 disadvantages of in vivo

1) would take days or weeks to produce the same quantity of DNA as in vitro. 2)  It involves culturing the bacteria or vector which is often a complex and long procedure

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Give 3 ways genetically engineered organisms can benefit humans..

1)Agriculture (higher yields, more nutritious, pest resistant, weather resistant) 2)Industry (processes often use enzymes-can be produced in large quantities for less money) 3)Medicine (drugs/vaccines made quickly + cheaply in large quantities)

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Give a real-life example of... a) use of GM organisms in agriculture

Golden rice- contains a gene from a daffodil plant, and one from soil bacterium. Enables plant to produce beta-carotene, which is used by our bodies to produce vitamin A. (used in areas with a deficiency e.g. South Asia

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b) Industry

Chymosin- an enzyme used in cheese-making. Made from rennet before (a substance produced in stomach of cows), but can now be made from GE organisms. --> in large quantities, cheaply and without killing any cows!

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c) Medicine

e.g. insulin- used to treat type 1 diabetes and used to come from animal's pancreases (mainly pigs). Insulin was often rejected by human immune system, but now GE microorganisms using cloned human insulin gene can make human insulin

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Give 3 advantages of using recombinant DNA technology in this way

1) can reduce famine and malnutrition 2) could make drugs more available to people e.g. in areas where refrigeration isn't available. 3) medicines produced more cheaply, so more people can afford them

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Give 4 risks of using recombinant DNA technology in this way (3 on this card, 1 on other)

1)could damage environment e.g. reduction of biodiversity 2) possibility of 'superweeds'- resistant to herbicides (would occur if wild plants interbred with transformed crops) 3) technology could be used unethically e.g. designer babies

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4th risk...

Without proper labelling, people may feel they won't have a choice about whether to consume GM food

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What does Genetic Therapy involve?

Altering defective genes inside cells to treat genetic disorders and cancer.

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If the disorder is caused by a dominant allele, gene therapy aims to...

...silence the dominant allele

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If the disorder is caused by recessive allele, gene therapy aims to...

add a functioning dominant allele (gene supplementation- faulty gene is replaced with a healthy one)

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How is the 'new' allele inserted inside the cell?

using vectors e.g. viruses, plasmids or liposomes

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What are the two different types of gene therapy?

1) somatic 2) germ line

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What is somatic therapy?

It involves altering the alleles in body cells, particularly those most affected by the disorder. - disease can still be inherited by offspring

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What is germ line therapy?

Involves altering alleles in gametes. Every cell of any offspring produced from these cells will be affected by the gene therapy and they won't suffer from the disease (currently illegal in humans)

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Give 3 advantages of gene therapy.

1) could prolong lives 2) could give people a better quality of life 3) could decrease the number of people that suffer (germ-line therapy only)

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Give 7 disadvantages of gene therapy (4 on this card)

1) effects of treatment may be short-lived (somatic) 2) Patient might have to undergo multiple treatments (somatic) 3) Might be difficult to get the allele into specific body cells 4) Body could start an immune response against vectors

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3 other disadvantages of gene therapy...

5) allele could be inserted into the wrong place in DNA- could cause further problems e.g. cancer 6) inserted allele could be over-expressed, producing too much of the missing protein 7) Disorders caused by multiple genes would be difficult to treat.

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What is a DNA probe?

a short, single-stranded section of DNA that has some sort of label attached that makes it easily identifiable.

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The two most commonly used probes are:

1)Radioactively labeled probes- made up of nucleotides with an isotope of P- identified using a photographic plate that is exposed by radioactivity 2) Fluorescently labelled probes- emit light under certain conditions

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Describe how DNA probes can be used to identify single genes in a sample of DNA (4 steps) -give first 2 steps

1)DNA probe is made that has bases complementary to the portion of DNA sequence that makes up part of the gene whose position we want to find 2)The DNA being tested is treated to separate its two strands

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Give last 2 steps..

3) The separated DNA strands are mixed with the probes, which binds to the complementary bases on one of the strands→DNA hybridization 4) 4. The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits

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What does a DNA microarray do? What is it?

Screens lots of genes at once. 1. A glass slide with microscopic spots of different DNA probes attached to it in row.

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Give 4 steps of using DNA microarray (2 on this slide, 2 on next)

1. A sample of labeled human DNA is washed over the array 2. If the labeled human DNA contains any DNA sequences that match any probes, it will stick to the array

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Next two steps...

3.The array is washed, to remove any labeled DNA that hasn’t stuck 4. Array then visualised under UV light- any attached DNA will fluoresce

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What is the sanger method?

A method used to sequence the exact order of nucleotides in a section of DNA

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How does the sanger method work?

Uses modified nucleotides that cant attach to the next base in the sequence when they are being joined together. • Hence act as terminators, ending the synthesis of DNA strand • 4 different terminator nucleotides are used, each with 1 of the 4 bases

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Describe the 4 step process. (1st step)

1) set up four test tubes, each containing: DNA polymerase, DNA primer, free nucleotides, fluorescently labelled modified nucleotide

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(2nd + 3rd step)

2) The tubes undergo PCR Many strands of DNA are produced of different lengths. (each 1 terminates at different points depending on where modified nucleotide was added) 3) Separation of DNA fragments by electrophoresis and visualised under UV light

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4th step

4) Complementary base sequence read from the gel - by reading the bands from the bottom of the gel to the top, you can build up DNA sequence one base at a time.

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Explain how the gel can be read

The smallest nucleotide (e.g. 1 base) is at the bottom of the gel (moved the furthest). Each band after this represents one more base added. Hence by reading the bands from the bottom to top, you can build up DNA sequence 1 base at a time

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For up to how many bases on a DNA fragment does this method work for?

Up to 500 base

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So what do we do about larger genes and whole genomes?

cut into smaller fragments using restriction endonucleases and each fragment sequenced. - then must piece sequenced fragments together using restriction mapping

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What is restriction mapping used for?

to put smaller sections of DNA that have been sequenced back into the correct order…

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Describe 4 step process of restriction mapping (first 3 steps)

1.Different restriction enzymes are used to cut labeled DNA into fragments 2. The DNA fragments are then separated by gel electrophoresis 3. The size of the fragments produced is used to determine the relative locations of cut sites

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4th step

4. A restriction map of the original DNA is made – i.e. a diagram of the piece of DNA showing the different cut sites, and hence where the recognition sites of the restriction enzymes used are found.

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What is gel electrophoresis?

When DNA fragments are placed on an agar gel and a voltage is applied across it -resistance of the gel means the larger fragments, the more slowly they move -over a fixed period, smaller fragments move further than the larger ones.

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In the past, these technologies were labour intensive, expensive and could only be done on a small-scale. Now...

...these techniques are often automated, more cost-effective and can be done on a large scale

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What can genetic screening be used to do?

locate mutated genes

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Some mutations can produce genes that are useful in some situations, and not in others. For example..?

Sickle cell anaemia.

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What is sickle cell anaemia?

A recessive genetic disorder caused by a mutation in the haemoglobin gene.

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What does the mutation cause?

An altered haemoglobin protein can be produced, which makes red blood cells sickle-shaped • The sickle red blood cells block capillaries and restrict blood flow, causing organ damage and periods of acute pain

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How can the mutation be advantageous?

Sickle-cell carriers are partially protected from malaria -Advantageous in areas where malaria is common, which has hence increased its frequency within the gene pool

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How can it not be advantageous in other situations?

it also increases the likelihood of people in those areas inheriting two copies of the sickle-cell allele, which means more people will suffer from the disease in those areas

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What is genetic counselling?

the advising of patients and their relatives about the risks of genetic disorders.

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What three things does genetic counselling involve?

1) advising people about screening + explaining the results 2) Screening can identify the carrier of a gene, the type of mutated gene they’re carrying and the most effective treatment. 3) advise patient on options on prevention/treatment available

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In the case of oncogenes, screening can help to detect what 2 things?

1) mutations, which can determine the type of cancer that the patient has and hence the most effective drug/radiotherapy to use 2) • a single cancel cell among millions of normal cells, identifying those at risk of relapse

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What does genetic fingerprinting rely upon?

the fact that the genome of any organism contains many repetitive, non-coding bases of DNA (introns). Introns contain repetitive sequences of DNA called core sequences, for which the number and length varies in every individual (except twins).

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What are the five stages in making a genetic fingerprint?

1.Extraction 2.Digestion 3.Separation 4.Hybridisation 5.Development

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Describe Extraction...

DNA is extracted from sample such as a drop of blood/hair root, by separating it from the rest of the cell. The quantity of DNA is increased by PCR.

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Describe Digestion...

The DNA is cut into fragments using restriction endonucleases, which are chosen for their ability to cut close to, but not within, groups of core sequences

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Describe Separation... (3 steps)

1)DNA fragments are separated according to size by gel electrophoresis under influence of an electrical voltage. 2)gel is immersed in alkali to separate double strands into single strands. 3)The single strands are transferred onto a nylon membrane

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Describe the process of southern blotting (the transfer of single strands on nylon membrane-2 of 4 steps)

1)Thin nylon membrane laid over gel 2)Membrane covered with several sheets of absorbent paper- draw up liquid containing DNA by capillary action

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step 3 and 4 of southern blotting...

3)Transfers DNA fragments to the nylon membrane in precisely same relative positions that they occupied on the gel 4)• The DNA fragments are then fixed to the membrane using UV light

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Describe Hybridisation...

Radioactive (or fluorescent) probes are used to bind with the core sequences. The probes have base sequences complementary to the core sequences, and bind to them under specific conditions of temperature and pH.

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Describe Development... (3 steps)

1)An X-ray film is put over the nylon membrane 2)film exposed by the radiation from radioactive probes. (if using fluorescent probes, positions located visually) 3)points correspond to DNA fragments' position as separated during electrophoresis

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And so?

A series of bars is revealed, with a pattern that is unique to every individual

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Describe Interpretation...

DNA from the suspect is compared to the DNA from the scene (e.g. from blood, hair, semen etc.)If samples look similar, they're checked on a computer, which can measure exact lengths of the DNA fragments + calculate the odds of someone else with same

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Give four other uses

1)Paternity issues- bars made on band should match that of parent 2) measuring variety in population- closer bands are, less variety 3)prevent undesirable inbreeding of animals 4)treatment of huntingdon's disease

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How is it used to treat huntingdon's?

Patient's DNA fingerprint is compared to others with the disease, to find out what symptoms they are likely to have, and when they will develop.

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Why might the matching of a genetic fingerprint not necessarily mean the suspect committed the crime? (3 reasons)

1)suspect'** DNA may've been left there at a innocent time 2)The DNA is from a very close relative. 3)The DNA has been contaminated after the event, either by the suspect's DNA, or by chemicals that affect the way restriction endonuclease cut the DNA

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Get Revising

Unit 5- Genes and DNA (Ch14-16)

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