4- anaylsis of DNA invitro

what is PCR

The polymerase chain reaction is a technique used to copy (or amplify) DNA samples

what are the 3 main stages of PCR

1. separation of DNA strands

2. addition of primers

3. synthesis of DNA

1. separation of DNA strands

the DNA fragment is heated to 95c to break the hydrogen bonds to separate the 2 strands

2. addition of primers

we add can add primers and lower the temp to a range of 65 to 55 to allow hydrogen bonds to form so the primer can anneal to the DNA

what is a primer and what’s there jobs

a primer is a short sequence of single stranded DNA bases they annel to a complimentary sight creating a small section of double stranded DNA as DNA polymerase can only extend / bind to double standed DNA

As primers only binding to specific sights what does this mean

they can only bind to a specific area so only this area can be replicated so were able to amplify specific genes

synthesis of DNA

due to the double bonded region DNA polymerase can bind and creates phosodiester bonds to join up the molecule.

what is the special type of polymerase

TAQ

what is TAQ polymerase

thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature

what’s its optimum temp

72

if there are 5 rounds of PCR how many DNA molecules do you have

you start with 2 strands so do 2 to the power of 5

electrophoresis

This is able to separate the DNA into strands to compare how similar it is

describe the process of electrophoresis

The DNA samples are placed into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and longer lengths of DNA are retarded by the gel so move more slowly than shorter lengths. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time.

how can we make the DNA visible

can be stained with dyes or radioactive tracers and then use autoradiography

what is autoradiography

Ordinary photographic film (sometimes called X-ray film) is placed on top of the finished gel in the dark for a few hours, and the radiation from any radioactive DNA on the gel exposes the film. When the film is developed the position of the DNA shows up as dark bands on the film. This method is extremely sensitive.

transcription factors

these bind to the promoter point to cause RNA polymerase to bind tightly to the gene.

what is the southern blot

A Southern blot is used to detect a specific target sequence in samples of DNA using a DNA probe.

What’s a DNA probe

a short length of single-stranded DNA with a radioactive or fluorescent label attached.

what does the probe do and what’s this process called

The probe will anneal to any fragments of DNA containing the complementary sequence, forming regions of double-stranded hybrid DNA this process is called hybridisation.

What’s done before hybridisation

DNA is extracted from the source cell and amplified by PCR to make enough DNA for the hybridisation

step 1 of hybridisation

The DNA samples are then digested by a restriction enzyme into many small fragments, and the fragments separated on an electrophoresis gel.

step 2

The gel is placed in an alkali solution, which breaks the hydrogen bonds between the DNA bases causing the stands to separate. A thin sheet of nylon is placed on top of the gel. The alkali solution is then drawn up through the gel to a stack of paper towels by capillary action, bringing the DNA with it. The DNA sticks to the nylon membrane.

step 3

The nylon sheet is separated from the gel, placed in a plastic bag containing a solution of probes label with either dyes or radioactivity and mixed. The probes will anneal to DNA fragments in the nylon membrane that have a complementary sequence, forming hybrid DNA molecules stuck to the nylon sheet

how is the DNA detected step 4

dyes can then be mad visible and then radioactivity can be detected by using autoradiography film

what is sanger/ DNA sequencing

This means reading the base sequence of a length of DNA.