BIOTECHNOLOGY

What is PCR?

Polymerase Chain Reaction - used by researchers to create vast quantities of DNA identical to trace samples. Essentially, it copies DNA.

Components of PCR

• DNA sample

• Thermal Cycler

• Primer

• Taq Polymerase (DNA polymerase)

• Free nucleotides

Steps of PCR

1. Denaturation: DNA strands are separated due to high temperatures (94 - 99^oC), breaking the hydrogen bonds and unwinding the DNA helix.

2. Annealing: the temperature is lowered (50 - 60^oC) and primers, free nucleotides & Taq polymerase is added. The primers bind to the sequence of DNA at the start of the gene being tested. This signals the free-floating nucleotides and Taq polymerase where to start binding.

3. Extension: The temperature is once again raised (approx. 70^oC) and Taq polymerase binds to the DNA sequence. This binds the new, complimentary nucleotides to the target gene. This extends the nucleotide chain and creates a new strand of DNA.

4. Steps 1 - 3 are repeated for 25+ cycles until enough copies of the target DNA have been produced.

Why do we use Taq polymerase instead of DNA polymerase?

Usually, incredibly high temperature kills enzymes (e.g. DNA polymerase) but Taq polymerase is able to withstand the high temperatures of PCR and complete the same function of DNA polymerase.

What is Gel electrophoresis?

a technique used to separate DNA fragments (or RNA and proteins) based on their size and charge.

How are DNA fragments separated? Why?

DNA fragments are separated based on size only. This is due to all DNA molecules having the same amount of charge per mass.

What is a gel?

a slab of jelly like material.

What is gel made out of?

Made from the polysaccharide called agarose, which comes as dry, powdered flakes.

How is the gel made?

The agarose is heated in a buffer (water with some salts in it) and allowed to cool, forming a solid, slightly squishy gel. The gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.

How is gel electrophoresis constructed?

The gel has pocket-like indentations called wells where the DNA sample is place. However, before this the gel must be placed in a gel box, which is filled with a buffer solution (which just barely covers the gel) that can conduct a current. The end near the wells is hooked to a negative electrode and the other end is connected to a positive electrode (both are connected to a power source).

What is one of the wells reserved for?

DNA ladder - a standard reference that contains DNA fragments of known lengths.

How do DNA fragments move through the gel?

Power source is turned on and a current begins to flow through the gel. The DNA (which is negatively charged) moves towards the positive pole.

Why is DNA negative?

The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone.

How does the DNA separated?

Shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones, which shorter pieces being close to the positive end while larger pieces will remain near the wells.

How do we visualize the DNA fragments?

The gel is stained with a DNA - binding dye and placed under UV light, leading to the DNA fragments to glow, allowing us to see the DNA present at different locations along the length of the gel.

How is the length of the DNA bands measured?

bp - how many base pairs long the DNA fragment is.

What is a band?

a well-defined “line” of DNA on a gel

What do bands contain?

Many DNA fragments of the same size that have all travelled as a group to the same position.

What can we determine from comparing the bands in a sample to the DNA ladder?

we can determine their approximate sizes.

What is DNA sequencing?

is the determination of the precise order of nucleotides in a sample of DNA.

What method is frequently used in DNA sequencing?

The Sanger Method

What are the different nitrogenous bases?

Adenine, Cytosine, guanine and thymine (uracil in RNA)

What is the more correct name for nucleotides?

Deoxynucleotide triphosphates

What happens when DNA forms?

• Each nucleotide loses two phosphate groups

• The sugar molecule loses a hydrogen atom from the hydroxyl group (OH) when it bonds to the phosphate group of an adjacent nucleotide.

What is the name of the synthetic nucleotides from Sanger’s method? what do they lack?

Dideoxyribonucleotide triphosphates/dideoxyribonucleotides (ddNTP’s)

They lack the OH group in the sugar molecule (3’ end)

What do synthetic nucleotides do?

Stops the elongation of the sequence as there is no OH group for the next nucleotide to attach to.

Applications of DNA sequencing

• identify mutations

• compare the DNA of different organisms

• identify inherited disorders

• identify some forms of cancer

• used for maternity and paternity tests

• compare species in order to track evolutionary changes

Steps for DNA sequencing

1. DNA is heated and separated into two strands (denaturation)

2. A mixture is prepared containing the DNA strand, a primer, DNA polymerase, free nucleotides and termination nucleotides (altered nucleotides)

3. Altered nucleotides are missing the OH group which prevents nucleotides from being added

4. The primer binds to the start of the DNA template

5. DNA polymerase assembles nucleotides complementary to the template strand

6. If an altered nucleotide is added, nucleotide assembly stops creating different lengths of DNA

7. The new DNA strands are separated by gel electrophoresis where the shortest sequences will travel the furthest creating a banding pattern.

8. Fluorescent markers on altered nucleotides are then read by a laser, and information is relayed to a computer.

What are the colours of the different dideoxyribonucleotides?

Guanine - Yellow/black/orange

Thymine - Red

Adenine - Green

Cytosine - Blue

Limitations of Sanger’s method

• Expensive

• Inefficient for larger - scale projects.

Features of next gen sequencing

• Highly parallel: many sequencing reactions take place at the same time

• Micro scale: reactions are tiny, and many can be done at once on a chip

• Fast: because reactions are done in parallel, results are ready much faster

• Low - cost: sequencing a genome is cheaper than with sanger sequencing

• Shorter length: reads typically range from 50 - 700 nucleotides in length