A Level Summary Of Gene Technologies - Basic

Front 1

Gene technologies are all the techniques used to study genes and their function. These techniques include...

Back 1

1) the polymerase chain reaction (PCR)

2) cutting out DNA fragments using restriction enzymes

3) gel electrophoresis

4) finding specific sequences of DNA using DNA probes

Front 2

What is the polymerase chain reaction?

Back 2

- artificial DNA replication

- generates multiple copies of the DNA sample

- can only copy pieces of DNA a few 100 bases long

- needs a primer (single-stranded DNA 10-20 bases long) to allow DNA polymerase to bind.

Front 3

what are primers?

Back 3

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

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What enzyme is used in PCR?

Back 4

DNA Taq polymerase

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What are the 3 steps of PCR?

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1) Hydrogen bond breaking - separation of DNA strands by heating to 95 degrees.

2) primer annealing - mixture is cooled to between 45-62 degrees so that the primers can bind to the strands

3) extension - the reaction mixture is heated to 72 degrees so DNA Taq polymerase can work. DNA polymerase extends the primers using free nucleotides. Complementary base pairing means complementary strands are formed.

(the whole process can be repeated many times so that the amount of DNA increases expotentially x2,x4,x8 and so on)

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what does gel electrophoresis do?

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separates DNA fragments according to size

(accurate to one base difference in length)

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how is the gel electrophoresis apparatus set up?

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1) formation of the gel plate

- Agarose powder is dissolved and poured into a gel mould

- A comb is inserted into the gel and left to set

2) setting up the tank

- the gel, once set, is placed into the electrophoresis tank

- a buffer solution is added completely covering the gel plate

-the buffer solution provides free electrons which can produce a current when attached to the electrodes

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Describe the electrophoresis process...

(treating and loading DNA, electrophoresis process and the results)

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1) treating and loading the DNA - DNA sample is treated with restriction enzymes to cut it into fragments. DNA is mixed with a dye and placed into wells in the agar gel plate at the anode end (negative end)

2) electrophoresis process- the DNA is negatively charged due to the phosphate groups and so when the current flows it will be pulled to the cathode (positive electrode). Shorter pieces of DNA will travel further up the gel compared to longer pieces)

3) Results - The position of the fragments can be shown by using a dye that stains DNA.

Front 9

what are restriction enzymes?

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-restriction enzymes (endonucleases) are isolated from bacteria and they cut at specific palindromic sequences of between 4-6 bases long.

They hydrolyse the sugar-phosphate back bone of the DNA molecule

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what is a palindromic sequence?

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antiparallel base pairs that read the same in opposite directions.

(different restriction enzymes cut at different specific recognition sequences, because the shape of the palindromic sequence is complementary to an enzymes active site)

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what are DNA probes?

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DNA probes are short single stranded pieces of DNA (50-80 bases long)

They are complementary in base sequence to the piece of DNA under investigation - used to identify fragments containing specific sequences.

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what are DNA probes used to locate?

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1) a desired gene or genetic engineering

2) genes for comparisons between genomes

3) identify the presence or absence of alleles

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What is the DNA probe labelled with?

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either

1) radioactive isotope groups (visible when exposed to photographic film e.g P32)

2) Fluorescent markers that are visible under UV light

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DNA sequencing (sequencing both genes and genomes) used a variety of technologies.

List the technologies used to sequence DNA...

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This technology uses both PCR and gel electrophoresis. The reaction mixture contains:

Taq polymerase, DNA primers, DNA sample strand, DNA nucleotides, DNA nucleotide with fluorescence markers (chain terminating nucleotide)

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What is the first step in DNA sequencing?

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Step 1 = PCR and terminating nucleotides

The reaction mixture undergoes PCR, which produces many strands of DNA. The strands are different lengths because each one terminates at a different point, depending on where the modified nucleotide was added (chain terminating nucleotide- dideoxy nucleotide)

The Taq polymerase can attach a normal free or chain terminating fluorescent tagged DNA molecule.

It is random which binds in each cycle and with enough cycles DNA fragments will be synthesised for every possible length of DNA.

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What is the second step in DNA sequencing?

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step = gel electrophoresis

DNA fragments all separate according to length. If the fragments are allowed to run off the gel plate they break a laser beam and the colour of the fluorescent marker is recorded.

The first fragment of DNA off the gel will be 1 base in length and represent the first base in the sequence. The next off the gel is two bases long and the colour identifies the second base. This continues until the whole DNA sample has been sequenced.

This is known as the chain termination method.

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The chain termination method can only be used for DNA fragments up to about 750 base pairs long. So if you want to sequence the entire genome (all the DNA of an organism) you need to chop it into small pieces first.

Smaller pieces are sequenced and then put back in order to give the sequence of the whole organism (genome). Here is how it is done...

Back 17

Genome sequencing:

1) A genome is cut into smaller fragements (about 100,000 base pairs long) using restriction enzymes

2) the fragments are inserted into BAC (bacterial artificial chromosomes) - man made plasmids. Each fragment is inserted into a different BAC.

3) The BAC are then inserted into bacteria - each bacterium contains a BAC with a different DNA fragment

4) the bacteria divide, creating colonies of cloned (identical) cells that all contain a specific DNA fragment. Together the different colonies make a complete genomic DNA library

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

6) Each piece of DNA is sequenced, using the chain-termination method

7) pieces are put back in order to give the full sequence from the BAC (using powerful computer systems)

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what is recombinant DNA?

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DNA from 2 or more different sources joined together.

(the organism that receives the gene is referred to as the recipient and is known as being transgenic)

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How to make recombinant DNA part 1 - obtaining the gene of interest

This can be done by 3 methods.

List these 3 methods...

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1) method 1- it can be located using probes to locate the gene on DNA fragments. The gene can be cut from a DNA fragment using a restriction enzyme - produces sticky ends which are complementary to the vector.

2) if the DNA sequence is known, the gene can be sequenced. Sticky ends are added to the gene which will be complementary to the ones formed when the vector is cut.

3) isolate mRNA from the transcribed gene and use the enzyme reverse transcriptase which makes a single stranded DNA copy of the mRNA strand. Then add DNA polymerase to make it double stranded, sticky ends are added which will be complementary to the ones formed when the vector is cut.

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part 2- inserting the gene into a copy of the vector/making recombinant DNA...

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the next step is to insert the DNA fragment into vector DNA- a vector is something that's used to transfer DNA into the cell.

1) the vector DNA is isolated

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

3) the vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sugar-phosphate backbone of the two bits of DNA ---> seals the recombinant DNA.

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To identify whether the vector (plasmid) has been taken up, we use marker genes. Marker genes are sequences of DNA that are associated with particular traits. The markers can be inserted into the vectors at the same time as the desired gene.

Give an example of a marker gene...

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- inserting antibiotic resistance genes into the plasmid

- if the cells are grown on agar plates containing the specific antibiotic, only transformed cells that have the marker gene will survive and grow.

-therefore if a bacterium can grow on that antibiotic, it indicates that the recombinant plasmid is present in the bacterium.