6.1.3 manipulating genomes

What is a restriction enzyme

Enzyme that cuts DNA at specific recognition sites which produces DNA fragments

Where are Restriction enzymes normally found

Bacteria

what is a recognition site ?

Specific base sequence

Why are recognition sites palindromic ?

Read the same backwards and forwards

What are the types of cuts made by restriction enzymes

Sticky ends

Blunt ends

what is the difference between sticky and blunt ends

Sticky ends → there’s a overhang of DNA bases

Why are restriction enzymes used on Non - coding genes?

There are no mutations in Non- coding genes base sequence → easier to predict results

Function of non- coding genes

Control transcription by switching genes on and off

what is the function of Gel electrophoresis

Separate the DNA fragments as DNA is invisible

Why does DNA move through the gel when an electric current is applied?

DNA contains PO₄³⁻ anion on nucleotide this is attracted to the Positive electrode

what is the colour of DNA

Colourless

Control variables when using enzyme

Use the same restriction enzyme

Function of the gel in gel electrophoresis

Creates a matrix mesh that keeps large DNA fragments at top ( cant travel through mesh) and small at the bottom

which DNA fragments move faster and further in Gel electrophoresis

Small fragments

Function of Buffer ( Salt) solution in gel electrophoresis

maintains charge helps the current move

why are DNA fragments / banding pattern different?

Different alleles → different specific recognition sites

Different number and sizes of DNA fragment are produced

DNA fingerprinting ( Image)

Limitations with gel electrophoresis

DNA fragments can get stuck in the gel

its not 100% could be related ( Identical twins)

Function of PCR in DNA fingerprinting

Used to amplify the DNA so there is enough to be analysed

DNA profiling method

Extract DNA

DNA fragments are placed into agarose gel

apply electric current

DNA moves to positive electrode

smaller fragments move further and faster

compare banding pattern with DNA sample to see if there similar.

Function of PCR

Amplify DNA fragment for further study or processing

What does PCR stand for

polymerase chain reaction

Uses of DNA profiling

Forensics

diagnosis of genetic disorders

What are the steps of PCR

Denaturation → separate double helix

Annealing → Primers bind to the start of the target DNA

Synthesis → Building the new strand

PCR what are primers

short pieces of DNA that are complimentary to the bases of the fragment

PCR what happens during denaturation?

DNA mixture heated to 95°C → breaks Hydrogen bonds

separates DNA strands

Why can many cycles of PCR happen without needing new enzymes

DNA polymerase doesn’t denature at high temp

PCR what happens at annealing stage

55°C temp of reaction mixture

allows primers to bind to strands

PCR what happens at the synthesis stage

72°C temp allows DNA polymerase to work

DNA polymerase joins DNA nucleotides to template strand using complimentary base pairings → forms 2 new complimentary strands

what type of DNA polymerase is used in PCR

Taq DNA polymerase → doesn’t denature at high temperatures

human does denature at 72°C

what molecules are required for PCR

 DNA template

primers

nucleotides

DNA polymerase

Limitations of PCR

Expensive

Takes a long time

what molecules are required for chain termination method

DNA polymerase

Primer

DNA sample

nucleotides

Fluorescently-labelled modified nucleotide / terminator base

Chain termination method first step

Mixture of required molecules is added to separate tubes

chain termination method second step

Tubes undergo PCR

strands produced are different lengths → modified nucleotide terminates the strand ( cant extend strand)

modified nucleotide is added on different points

why do fluorescently-labelled modified nucleotides terminate the strand

No bases can be added after

Hydrogen instead of hydroxyl group on carbon 3 so cant form phosphodiester bond with next nucleotide

methods to read data in DNA sequencing

Gel electrophoresis

Capillary sequencing

electrophoresis method in DNA sequencing

electrophoresis separate DNA fragments

shine under UV light

Read from top to bottom ( smaller fragments are near top / move furtherest)

How can DNA be sequenced ?

chain termination method

Advantages of using chain termination method for DNA sequencing ?

allows for massive parallel sequencing

sequence many DNA at same time