Common Manipulating Genome Techniques 2019

Front 1

What are some techniques used to study genes?

Back 1

•The P olymerase C hain R eaction

•Gel electrophoresis

•Cutting out DNA fragments

Front 2

What is PCR used for?

Back 2

To amplify a certain fragment of a gene to produce millions of copies

Front 3

What is the first stage of PCR?

Back 3

A reaction mixture containing the DNA sample, free nucleotides, primers and DNA polymerase is set up

Front 4

What are primers?

Back 4

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

Front 5

What happens to the mixture after it is set up?

Back 5

It is heated to 95°C to break the H bonds between the two strands of DNA

Front 6

Why can DNA polymerase be reused in multiple PCR cycles?

Back 6

Because it doesn't denature at 95°C , which is good because new enzymes don't have to be used everytime

Front 7

What happens to the mixture after it is heated?

Back 7

It is cooled to between 50 - 60 °C so the primers can anneal to the strands of DNA

Front 8

Once the primers have had a chance to anneal, what is done to the mixture?

Back 8

It is heated till 72°C which allows DNA polymerase to work

Front 9

What does DNA polymerase then do?

Back 9

It lines up free DNA nucleotides alongside each template strand, in complementary base pairings; means a new complementary strand is formed

Front 10

What is formed after 1 complete cycle of PCR?

Back 10

Two new copies of the fragment of DNA are formed

Front 11

What happens next in PCR once, 1 cycle is complete?

Back 11

The entire process starts again, the mixture is heated to 95°C again and this time, the both the DNA double helix break up and all 4 strands act as a template.

Front 12

The number of DNA is... after each PCR cycle

Back 12

Doubled

Front 13

What does electrophoresis do?

Back 13

Separates DNA fragments by size

Front 14

How is the equipment for electrophoresis set up?

Back 14

1) Agarose gel which has been poured into a gel tray and left to solidify is placed into a gel box/ tank

2) A row of wells is created on one end of the gel tray and that side is placed closest to the negative electrode in the gel box

3) A buffer solution is added to the reservoirs at either side of the gel box so the surface of the gel is covered in buffer solution

Hint 14

Front 15

How is the fragmented DNA solution prepared for electrophoresis?

Back 15

Loading dye is added to fragmented DNA sample; this helps the samples to sink to the bottom of the wells and make them easier to see.

Front 16

Once the DNA fragmented solution is made, what is done next?

Back 16

A set volume of the DNA sample is placed into the well with a micropippete, being careful to not pierce the bottom of the well but insuring the tip is in the buffer solution just above the opening of the well.

Front 17

After you've placed one sample o DNA into one well, what do you need to control for the addition of the the other samples?

Back 17

•Same volume of other DNA sample added into the well

•Same volume of loading dye was added to the same volume of sample

•Use a clean micropippete each time

Front 18

How is electrophoresis carried out?

Back 18

•Place the lid on the gel box; this connects the leads from the gel box to the power supply

•Turn on power supply and set to required voltage; an electrical current passes through the gel

•DNA fragments are -ve charge so move through gel towards the anode

•Smaller fragments move faster and travel further; this separates out the fragments by size

Front 19

What is done after electrophesis is allowed to take place?

Back 19

• Turn of power supply when the dye is about 2cm from the anode

•Remove gel tray and tip out any excess buffer solution

•Stain the DNA fragments then rinse the gel with water. The bands of different fragments will be visible

Front 20

What is the size of DNA fragments measured in?

Back 20

Bases

E.g. ATCC = 4 bases

1000 bases is 1kb

Front 21

What else can electrophoresis be carried out on?

Back 21

RNA fragments

Front 22

Why are proteins more difficult to separate by electrophoresis?

Back 22

Because they can be positively or negatively charged

Front 23

What can be done to overcome the charge issue of proteins?

Back 23

They can be mixed with chemicals that denature the proteins so they all have the same charge

Front 24

What uses does the electrophoresis of proteins have?

Back 24

Can be used to identify proteins in urine or blood samples, which could help diagnose a disease

Front 25

Apart from PCR what can be used to cut out DNA fragments from an organisms DNA?

Back 25

Restriction enzymes

Front 26

What are palindromic sequence of nucleotides?

Back 26

Consist of antiparallel base pairs that read the same in opposite directions

Front 27

What do restriction enzymes do?

Back 27

They recognise specific palindromic sequences, AKA recognition sequences and cut (digest) the DNA at these places

Front 28

How can restriction enzymes therefore be used to cut out DNA fragments?

Back 28

If the DNA fragments wanted, have recognition sequences on either side. Restriction enzymes can be used to separate the DNA fragments from the rest of the DNA

Front 29

Does a restriction enzymes cut out all recognition sequences?

Back 29

No, only those recognition sequences that have a complementary shape to the restriction enzymes active site

Front 30

Gj

Back 30

Check or I need to know examples of which restriction enzymes cut what

Front 31

What is the actual process of cutting out DNA fragments using restriction enzymes?

Back 31

Incubate the DNA sample with specific restriction enzyme which have a complimentary active site, which will cut the desired DNA fragments

Front 32

What reaction is the DNA fragments cut out by?

Back 32

Hydrolysis

Front 33

Once the DNA fragments is cut, what is left sometimes?

Back 33

Sticky ends; small tails of unpaired bases at each end of the fragment

Front 34

What can sticky ends be used for?

Back 34

To bind/ anneal the DNA fragments to another piece of DNA that also has sticky ends which have complementary sequence

Front 35

What technique is used to produce DNA profiles?

Back 35

Electrophoresis

Front 36

What is compared in different organisms to create a DNA profile

Back 36

•Some of an organism genome consists of repetitive, non-coding base sequences- these don't code for proteins and repeat over and over

•The number of times this is repeated is different In different people and so is the length of these sequences in nucleotides

Front 37

What is analysed using electrophoresis?

Back 37

The no. of times a sequence is repeated at different loci in a person's genome (and so the no. of nucleotides there) can be analysed to create a DNA profile

Front 38

Is DNA profile an accurate way of identifying an individual?

Back 38

The probabilty of two individuals having the same DNA profile is very low because the chance of two individuals having the same no. of repeat sequences at each locus in their DNA is very low

Front 39

How is DNA profiling conducted in forensic science?

Back 39

1) DNA is isolated from the sample

2) PCR is used to amplify areas with different repeat sequences; primers bind to either side of these repeat so the whole repeat is amplified

3) The products undergo electrophoresis and a DNA profile is made

Front 40

What will you see if the DNA profile matches?

Back 40

The band's of the different DNA fragments will match

The same number of bands will be present

Front 41

If the samples used for electrophoresis are from the same species...

Back 41

The more similar the pattern of bands

Front 42

How can DNA profiling be used for medical diagnosis? What can it be refered as?

Back 42

Can be used to analyse the risk of genetic disorders; the DNA profile in medical diagnosis can refer to a unique pattern of several alleles

Front 43

What enzymes is used to convert mRNA into a single stranded DNA? Is used to find what genes to isolate

Back 43

Reverse transcriptase