UNIT 3 - AOS 1 - CH 2.4 & 2.5 - PCR, Gel electropherosis

Question 1

Polymerase Chain Reaction - PCR

Answer 1

Amplifies a sample of DNA by creating additional copies
- Multi-steps process that involved thermal cycling
- Used by scientists whenever there is insufficient amount of a DNA sample for testing.

Question 2

Parts used in PCR

Answer 2

DNA SAMPLE - Provides template to produce copies of

PRIMERS - To bind to the single-stranded DNA & provide point in which DNA synthesis can be initiated & designate the sequence to be copied.

DNA (taq) Polymerase - To make multiple copies of the DNA strand y adding nucleotides (binds onto primer & moves along)

FREE NUCLEOTIDES - to be added to DNA polymerase (Taq polymerase in bacteria) to produce new RNA strand.

MIX BUFFER - Provide suitable chemical enviro for activity of DNA polymerase, by maintaining appropriate pH & providing any required salts.

PCR TUBE - Vessel for PCR reaction to occur.

Question 3

3 steps of PCR cycle

Answer 3

STEP 1 - DENATURATION
- dsDNA (double stranded DNA) heated to 90-95.C for 1 min
- Breaks hydrogen bonds between strands
- Results = 2x ssDNA
- Repeat ~35x

STEP 2 - ANNEALING
- ssDNA is cooled to approx. 50-55 .C for 2 mins
- Allows primers (forward & reverse) to complementary bind to 3’ end ssDNA

STEP 3 - EXTENSION
- DNA heated to 72 .C for 1 min
- Optimal temp DNA (Taq) Polymerase to bind to primers & begins synthesis of new complementary strand of DNA by adding free nucleotides.
- Results = 2x dsDNA molecules

Question 4

What is gel electrophoresis and its purpose?

Answer 4

-> Allows you to take DNA fragments prepared using restriction endonucleases or Polymerase chain reaction (PCR) & separate them based on their size.

PURPOSE:
- DNA analysis (fingerprinting)
- Forensic investigations (e.g., DNA profiling -> comparing DNA crime scene)
- Examining disease (is gene of interest present or not?)
- Paternal disputes (e.g., who is father?)

Question 5

Process of Gel electrophoresis (x4)

Answer 5

STEP ONE: DNA samples placed wells
- DNA ladder of known size is also loaded into gel (ladder gives us something to compare to)
- Gel -> made of agarose (has tiny pores) & immersed in a buffer solution

STEP TWO: Electric current applied between to negative & positive electrodes.
- DNA is negative so loaded at negative end and moves towards positive end (it is attracted to + )

STEP THREE: Separation of DNA based on size
- Smaller DNA fragments = move through pores easier & faster = further
- Larger = slower = less far

STEP FOUR: Visualising DNA
- DNA gel needs to be stained with dye so bonds can be visible under UV lamp.

Question 6

Applications of genetic testing - with PCR & Gel Electrophoresis

Answer 6

Genetic disorders = mutations

How is it diagnosed:
- PCR with specific primers
- Gel electrophoresis using-
. Standard ladder (determine band size)
. Negative control (e.g. healthy baby sample)
. Positive control (e.g. sample of mutated CFTR gene)

Question 7

Applications of DNA profiling - With PCR & Gel Electrophoresis

Answer 7

Analyse short tandem repeats (STRs) -> found in non-coding area (introns)
= Small sections of repeated nucleotides that vary in length
- Hundreds of variants of STRs can be found -> ^ mutation rate than coding regions of DNA (exons)

HOW:
1. PCR for an STR (specific primers used)
2. Gel Electrophoresis (separates by size)
3. Interpret gel (Heterozygous for that STR = 2 bands (2 alleles))
(Homozygous for that STR = 1 thick band)