Gene Technology Fingerprinting

Question 1

Polymerase Chain Reaction

Answer 1

Used to amplify target DNA sequences that are present in a DNA source

Mimics natural process of DNA replication

Question 2

what Does PCR require

Answer 2

•a DNA sample that includes the selected region for replication

•synthesis of primers - short strands DNA that are complimentary to seq at start of each strand of the region to be amplified

•DNA polymerase - extracted from thermophilic bacteria - thermostable

•Free Deoxyribonucleotides

Question 3

PCR steps

Answer 3

Mixture placed in a thermal cylinder

•DNA to be amplified is heated to 95° for 1 min - breaks H bonds between bases - separates strands

•Mixture cooled to 50-60° - allows primers to anneal to start of each strand of comp DNA region - primers prevent DNA strands rejoining - act as signals to polymerase enzymes to start adding nucleotides

•mixture heated to 72° for 1-2min and thermostable polymerase enzymes (at opt temp) copies each strand starting at primers

•process repeated with each cycle number DNA molecules doubled - exponential amplification

Question 5

Anneal

Answer 5

To bind complementary sequences of single stranded DNA by H bonding

e.g. primers or a probe is annealed to single stranded DNA

Question 6

Uses of PCR

Answer 6

Used by forensic scientists and archaeologists to study minute samples of DNA

May be used in Genetic Fingerprinting (DNA profiling)

Question 7

What DNA used for DNA Fingerprinting

Answer 7

Non-coding DNA (introns) between genes that is used in Genetic Fingerprinting

It contains variable nucleotide sequences that are similar in related individuals but distinct in unrelated individuals

Question 8

Satellite Repeat Sequences

Answer 8

Variable non-coding DNA known as satellite DNA and contains short DNA sequences which are repeated many times

•Minisatellites contain 20-50 base pairs - repeated from 50 to several hundred times

•Microsatellite contain 2-5 base pairs - repeated 5 to 15 times

Question 9

Microsatellite Repeat Sequences used for

Answer 9

Genetic markers in DNA fingerprinting since the pattern repeat sequences in any individual other than identical twin is unique

Same repeat regions occur at same positions on both chromosomes but number of repeats may differ

Question 10

Restriction Endonucleases

Answer 10

Restriction enzymes cut the sugar phosphate backbone of DNA at specific nucleotide sequences

Bacteria produce these enzymes to counter attack by viruses (bacteriophages) - do this by cutting bacteriophage DNA into smaller non-infectious fragments

Question 11

Type of Cuts in Restriction Nucleases

Answer 11

Blunt Ends - straight
Sticky Ends - staggered

Question 12

6 Steps of Using Restriction Enzymes and Probes

Answer 12

1. Extracting DNA from Sample
2. Using restriction enzymes to cut out repeated sequences
3. Using gel electrophoresis to separate DNA fragments
4. Transferring DNA fragments onto a nylon sheet
5. Attaching labelled probes
6. Detecting different repeat sequences

Question 13

DNA Probes

Answer 13

Used to locate a particular section of DNA

Consists of short length of single-stranded DNA with a specific nucleotide sequence

Probe anneals by base pairing to a complementary region of single stranded target DNA

Probe has fluorescent and radioactive tag - detection takes place using UV light for fluorescent or X-ray film for radioactive

Question 14

Gel Electrophoresis

Answer 14

DNA fragments placed in a well at the negative electrode end of a gel plate

An electric current is passed through the gel - since DNA negatively charged - fragments move to positive end

Smaller fragments move further

Question 15

Hybridise

Answer 15

Binding of single-stranded DNA with complementary single-stranded DNA from another source

Question 16

DNA Profiling Using PCR

Answer 16

•fluorescent DNA primers that will attach next to region containing MRSs are synthesised

•Labelled fragments of DNA separated using gel electrophoresis

•UK Forensic Science Service - completed for 10 diff microsatellites with add. biomarker for gender

•position of DNA fragments revealed as pattern of Fluorescent bands due to tags on DNA primers flanking microsatellite regions - detected by laser scanner

•Results displayed ina graph of fluorescence against fragment size - DNA profile

Question 17

Uses of Genetic Fingerprinting

Answer 17

Forensic Science
Settling paternity disputes
Establishing family relationships
Studying genetic diversity of species
Evolutionary relationship between taxonomic groups