Studying Genomes

Learning Outcome

Outline the steps involved in sequencing the genome of an organism.

Cloning the chromosome section• The chromosome is physically sheared into

sections.

• Existing approximate chromosome maps allow identification of the location that each section has come from

• The sections are incorporated into BACs (Bacterial Artificial Chromosomes)

• A BAC is a modified bacterial plasmid that incorporates the foreign DNA.

• The foreign DNA is flanked by STCs (Sequence Tag Connectors) that act as markers to identify the incorporated DNA

• The BAC is inserted into a bacterium (using electroporation) and the whole BAC is copied each time the bacteria reproduce giving numerous clones of the DNA being investigated. These cells are called clone libraries.

Sequencing the BAC section (step one)

• A number of copies of the BAC section are extracted from the cloned bacteria

• Next the DNA is treated with restriction enzymes or endonucleases

Restriction enzymes • Restriction enzymes work

by cutting the DNA at specific sequences, often in a staggered way, to leave sequences of unpaired bases called sticky ends.

• If the cut is not staggered it leaves blunt ends.

• DNA ligase is used to stick the DNA sugar phosphate back bones back together

• Hydrogen bonds between the complementary bases hold them together

Sequencing the BAC section (step two)• Once the fragments of

DNA have been produced using restriction enzymes these can be separated by using electrophoresis.

• When the fragments are put into the agarose gel with an electric current across, the smallest fragments move through the gel fastest.

Learning outcome

Outline how DNA fragments can be separated by size using electrophoresis.

Electrophoresis • DNA fragments are placed in wells in

the agarose gel at the cathode end

• Gel is immersed in buffer and an electric current passed through

• DNA has –ve charge due to many phosphoryl groups so is attracted to positive electrode (anode)

• Short DNA fragments diffuse faster through the gel.

• Position of fragments is revealed by using a dye that stains DNA

• Fragments can be lifted from gel using “Southern blotting”

• Fragments can be further sequenced using PCR and Chain Termination

• Or probed to see if they contain mutations

Electrophoresis sites to look at

• http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

• http://www.dnalc.org/resources/animations/gelelectrophoresis.html

Sequence or probe?

• Once the DNA fragments have been separated you can either:

• See if any of the fragments contains the DNA sequence you are looking for by using a probe

Or• Determine the sequence of bases

using chain termination• Both techniques need to be

preceded by use of the polymerase chain reaction PCR

Learning outcome

Outline how the polymerase chain reaction can be used to make multiple copies of

DNA fragments.

Polymerase Chain Reaction (PCR)• Remove double stranded DNA

from gel by southern blotting• Heat DNA to 95 degrees C to

separate strands• Add primers (short bits of DNA for

DNA polymerase to bind to)• Reduce temp to 55 degrees.

Primers anneal (bind to complementary bases)

• Raise temp to 72 degrees and add DNA polymerase

• DNA polymerase binds and makes new complementary DNA using free nucleotides

• Repeat the process to increase DNA exponentially

PCR websites to look at

• http://lifesciences.envmed.rochester.edu/movies/PCR_final.swf

• http://www.sumanasinc.com/webcontent/animations/content/pcr.html

• NB Make sure you understand why the DNA polymerase used for this process is called “Taq” polymerase

Learning outcome

Describe how DNA probes can be used to identify fragments containing specific

sequences.

The Action of DNA Probes

• Single stranded DNA from PCR or electrophoresis can be investigated using probes

• These are short single stranded pieces of DNA complementary to the piece of DNA being sought.

• The probe is labelled with a radio-active or fluorescent marker

• When copies of the probe are added to samples they will anneal to any complementary strands

• Fogging of photographic film or fluorescing under UV light shows areas where the DNA sequence being sought is present

Microarrays for Genetic Testing• Microarrays can be made with many

probes fixed to a “chip”• Probes are made that have

complementary sequences to those found in faulty alleles for genetic diseases

• The test DNA fragments are applied to the microarray

• If fragments anneal to the probes this indicates that the person’s DNA is carrying the faulty alleles and they may develop the genetic disease

• Analysis is by comparing test DNA with the annealing and fluorescence patterns of known fragments

Automated sequencing of DNAusing chain termination

(outline the steps involved in sequencing the genome of an organism)• This method uses dideoxynucleotide

triphosphates(ddNTPs) in addition to normal nucleotides. The ddNTPs have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs)

• Dideoxynucleotides are chain terminators as there is no OH group to contribute to the condensation reaction that is required for the next nucleotide to add to the chain.

• Terminators are added randomly to the growing chains giving a range of fragment lengths.

• These are then passed through an electrophoresis gel and sequenced by laser

Suggested websites with animations about Automated DNA sequencing

• http://www.pbs.org/wgbh/nova/genome/sequencer.html#

• http://www.dnalc.org/resources/animations/cycseq.html

• http://www.wiley.com//college/pratt/0471393878/student/animations/dna_sequencing/index.html

• All of these are excellent • The last of these websites goes through the whole process

step by step explaining each part and has review questions to check your understanding. I recommend it!

Stretch and ChallengeRNA interference

• The presence of double stranded RNA in a cell initiates the action of an enzyme called “Dicer”

• Dicer cuts the dsRNA up and makes small inhibitory RNA (siRNA)

• siRNA complexes with several proteins to produce RISC (RNAi silencing complex)

• RISC unwinds the double strand• siRNA then binds to the active

mRNA that should code for a polypeptide during translation

• “slicer” enzyme chops the mRNA and degrades its nucleotides

Using RNA interference as a contraceptive

• Prior to fertilisation mature ova make ZP3 protein that allows sperm to bind to and penetrate the membrane

• No ZP3 protein =no fertilisation• Introduce dsRNA with a section

complementary to mRNA for ZP3

• Dicer chops dsRNA to produce siRNA

• siRNA complexes to form RISC• RISC unwinds dsRNA• siRNA binds to mRNA for ZP3• Slicer chops and degrades

mRNA so no ZP3 is made and no fertilisation takes place!

• Outline how gene sequencing allows for genome wide comparisons between

individuals and species.

• See page 167 of your text book

• Make notes on it for yourself!

Questions!• Make sure you can answer all the

questions on pages 166 to 173!

• Learn the stages in electrophoresis techniques

• Make sure you could explain how PCR is used in forensic investigations

• Why isn’t the DNA polymerase used in PCR denatured at high temperatures?

A whole website dedicated to biology animations etc!

• http://bio-alive.com/animations/DNA.htm