DNA Lecture 4 Flashcards
Human chromosomes display a one base difference in every ________ bases, on average
Every individual has some differences
1000
Base differences may or may not result in difference in amino acid sequences of proteins because
Not all segments of DNA code for proteins
There is more than one codon for most amino acids
The DNA sequence changes (single base differences) between individuals can affect recognition sites for
Restriction enzymes
Restriction enzyme sites are randomly made and eliminated
If a specific base sequence is present, the restriction enzyme which recognizes it will cleave the DNA molecule at the appropriate site and generate fragments of a specific length. However, if a restriction site is absent due to a base difference, different DNA restriction fragment lengths are formed.
!
Base sequence differences between individuals in a population are called
polymorphism
The variation in DNA restriction fragments between individuals are known as
Restriction Fragment Length Polymorphisms (RFLP)
The analysis of individual differences in RFLPs
In theory, there is a unique DNA fingerprint for every individual, the ____________ fragment pattern for each individual is unique
DNA finger printing
Restriction Enzyme
Detection of RFLPs relies on a specialized hybridization technique called
Southern Blotting
In DNA fingerprinting, first the DNA must be
Then the DNA must be
Then the DNA fragments must be
Then the DNA must be ______ so that it is single stranded
isolated from the rest of the cellular material in the nucleus
cut into several fragments of different sizes with restriction enzymes
Sorted by size
Denatured by soaking the agarose gel in alkaline solution
After the DNA is denatured in fingerprinting, the DNA is blotted onto a nylon membrane. The strands of DNA bind to the positively charged groups in the nylon membrane because of..
The negative charge on the DNA bases
After blotting the DNA, the membrane is immersed in a solution of radiolabeled DNA probe.
The probe is a 32P labeled oligonucleotide whose sequence is complementary to a segment of known sequence of DNA.
The oligonucleotide with hybridize to the fragment that contains its
Complimentary Sequence
Once the probe is bound to the DNA in DNA fingerprinting, the result is washed and analyzed via autoradiography.
The fragment length is determined from the position on the gel compared to DNA markers of
Known Length
Usually DNA fragments resulting from DNA fingerprinting (aka restriction digest) for paternity testing are separately analyzed with several 32P labeled oligonucleotide probes. Combining the use of these makes the test extremely selective.
Each gene fragment analyzed has a different paternity index, from those a combined paternity index is reported. A probability is calculated
Many mutations either create a new restriction site in a genome or destroy a previously existing site. If a mutation occurs within or near a particular gene, the restriction fragmentation pattern will change.
This change can be determined by ______ with a sequence that lies in the same region of the genome
Southern blotting
This disease has an altered restriction pattern when DNA is probed for part of the gene for the beta-globin subunit, enabling prenatal diagnosis
Sickle cell disease
Other ex:
Cystic Fibrosis, Huntington’s, cancer, manic depression
In a classic example of RFLPs, the semen sample obtained from a rape and murder victim was compared with samples from the victim and two suspects. The samples were cleaved by ________ and separated by ___________ and processed by ________
The samples were cleaved by endonucleases and separated by gel electrophoresis and processed by the Southern blotting procedure
Name the two disadvantages of the Southern blotting method in a crime scene
Southern blot requires fresh DNA samples and larger amount of DNA than those usually found on a crime scene
The need for more sensitive DNA typing methods led to a focus on the _________ and on ______
polymerase chain reaction (PCR) and on STRs
Most genetic fingerprinting done today analyzes regions of our genome referred to as _________ that have highly variable repeating sequences
Minisatellites
The minisatellites have _______ most commonly 4 bases long that are repeated several times, such as (TAGA)m
Short Tandem Repeats (STRs)
____ is observed between the number of repeats (n) in each STR region
Strs represent about ____% of all human DNA
Polymorphism
3%
By analyzing the number of repeats in many minisatellites, a unique profile for each individual can be obtained.
_______ is readily applicable to STR analysis
Polymerase Chain Reaction (PCR)
This procedure makes it possible to isolate and analyze the DNA in a very small tissue sample (e.g. one human hair, a drop of blood, a small semen sample, or samples that are many years old)
With the increased sensitivity, forensic scientists focused on STRs rather than RFLPs
PCR
In this first step of PCR, DNA strands are separated by
In the second step, strands are annealed to an excess of short DNA _____ that flank the region to be amplified
New DNA is synthesize by ____
heating
Primers
Polymerization
The DNA sequences flanking STRs are unique to each type of STR and identical in all humans
! Not really sure what this means
PCR primers are targeted to the unique flanking DNA and designed to amplify across the STR
The length of the PCR product is the length of the ____ in that sample
STR
How many products are there from one person in PCR? Why?
Are they similar or different?
Two, one for each chromosome from each parent
Frequently different
The resulting products from PCR are subjected to think capillary _____. The resulting fluorescent bands are scanned by a laser to generate a series of peaks.
The length of the minisatellite and the number of repeats is determined by comparing the capillary gel elution time of the minisatellite DNA to the elution time of DNA strands of known length
PAGE
Standardization is required for any forensic analysis. The US forensic STR standard was called the ____ and was established in 1998
Combine DNA Index System (CODIS
It is based on 13 well studied STR loci. These must be present in any DNA typing experiment carried out in the US
CODIS
The ______ gene, present on human sex chromosomes, is also used as a marker in experiments in addition to the 13 STR loci
It has slightly different flanking on X and Y chromosomes. Different sized products are generated which can reveal the _______ of the DNA donor
amelogen
gender
The complete sequence of the human genome (3 billion base pairs) was published in April 2003. Six nations were involved in this collaborative effort.
Human Genome Project
In the Human Genome Project, research teams first generated a physical map of the human genome. Then, individual clones were sequenced by shotgun sequencing protocols. Researchers used powerful, new automated sequencers to sequence random segments on a given clone.
The sequence of the entire clone was assembled by identification of
Overlaps
A separate commercial initiative sequenced the entire genome by shotgun
The genomes of many organisms have been sequenced and are available in public data bases
cloning
We have only _____ to ______ genes
Only ____ to _____ % of our DNA encodes proteins
30,000 to 35,000
1.1-1.4%
