DNA sequencing

DNA Sequencing


Determines a sequence of base pairs in a

DNA fragment.


The DNA sequencing technique is very time

consuming and can be done on only short

fragments.


Initially the DNA fragment is copied.


Then placed in test tubes.


Once in a test tube a chain terminating

nucleotide is added.

A chain terminating nucleotide is a substance that

cuts the DNA fragment into smaller segments.


The last nucleotide is known.


There are four different chain terminating

nucleotides 🡪 adenine, guanine, thymine and

cytosine.


Each test tube has a different chain terminating

nucleotide therefore each DNA fragment ends in

that known nucleotide.


DNA Sequencing cont…

Once the fragments are cut, they are loaded onto

a sequencing gel.


The fragments will order themselves on the

sequencing gel.


The shortest strands of DNA travel the shortest

and they are found at the bottom of the gel

sequencing page.


Once each test tube of fragments have been

sequenced, they are put together to form the

complete sequence.



Electrophoresis


Sequencing gel is a

matrix containing small

spaces.


The DNA fragments

are charged.


The charged DNA

moves because of an

electric field.


Arranged based on

length.


DNA Sequencing cont…


Was used to complete the Human genome

project.


Short fragments at a time, one gene, one

chromosome at a time.


The aim of the project was to gain

knowledge of how to treat genetic

disorders.


Enzymes and Recombinant DNA

Enzymes are used to isolate fragments of

DNA.


Once the DNA has been isolated it can be

modified.


This modified DNA or original fragment can

be transferred to another organism.

The most common Enzymes:


Restriction Endonucleases 🡪 restriction

enzymes


Methylases


DNA Ligase


Taq DNA Polymerase

Restriction Endonucleases


Restriction Enzymes


Biological Scissors


An enzyme that cuts DNA at specific

sequences. 🡪 recognition sites.


Each type of restriction enzyme has a

specific recognition site.


Recognition sites are 4-8 nitrogen base

sequences.

Uniquely they are palindromic 🡪 each

strand has the same sequence as it is read

from 5’ to 3’.


Table 2 p. 680


Sticky Ends 🡪 the restriction enzyme cuts

the DNA molecule between the same

nucleotides and produces an overhang.

Sticky End


Enzyme EcoRI


Recognition site GAATTC


CTTAAG

Restriction Endonucleases


Blunt ends 🡪 occur when restriction

enzymes cut between the same nucleotides

and no overhang is created. (SmaI)


Sticky ends are liked by biologists because

of their ease of inserting new DNA to the

sticky end.


Restriction enzymes are chosen to cut in

areas not affected by the particular gene to

be isolated.

Methylases


Protect a gene fragment from being cut in a

certain place.


Can be added to restriction enzyme

recognition sites.


By adding methylases to a gene location

biologists can direct a restriction enzyme to

cut where they desire.


Methylase is added to

stop EcoRI from

cutting at this

recognition site.

DNA Ligase


Is an enzyme that can repair an join

sequences of DNA.


Biological glue.


DNA ligase is used in DNA Replication and

creating recombinant DNA.


DNA ligase will join the DNA strands

together after a sticky end is created and

matched up with a complementary DNA

sequence.

DNA Ligase glue.


Allows for the sticky

ends to be formed with

the compliment.


Hydrogen bonds can

form.

Taq DNA Polymerase


Polymerase Chain Reaction 🡪technique


In the process millions of copies of DNA

fragments are made.


Taq DNA Polymerase is an enzyme key to

PCR.


Taq DNA Polymerase produces DNA during

replication.


Process 🡪 Figure 7 p. 682

Heat breaks the

hydrogen bonds.


DNA becomes

templates.


Cooling and primers

help to form the new

complimentary strands.


Heating and cooling

cycles.

Transformation


Is any process when DNA is incorporated

into the genome (DNA) of a cell.


That cell will have altered DNA and will

produce new cells with the incorporated

DNA 🡪 as if it were its own.


The organism now is called a transgenic.



DNA Sequencing and Key Concepts (80/20 Summary)

In DNA sequencing, the essential functions can be captured succinctly:

  • Basic Definition: DNA sequencing determines the order of base pairs in DNA fragments, allowing for the identification of genetic information.

  • Process Overview:

    • Copy the DNA fragment.

    • Insert in test tubes with chain-terminating nucleotides (adenine, guanine, thymine, cytosine).

    • DNA fragments cut and separated on a sequencing gel; shortest strands move the furthest.

  • Electrophoresis: Charged DNA moves through a gel due to an electric field, arranged by length.

  • Human Genome Project: Used DNA sequencing to improve understanding of and treatment for genetic disorders.

  • Key Enzymes:

    • Restriction Endonucleases (Biological Scissors): Cut DNA at specific sequences.

    • DNA Ligase (Biological Glue): Joins DNA fragments together.

    • Taq DNA Polymerase: Essential for the Polymerase Chain Reaction, making millions of DNA copies.

  • Transformation: Process of incorporating new DNA into a cell's genome, creating transgenic organisms.